Note: From Sept 2009, articles are marked as a Premier Publication by the symbol P using practice similar to the BNL NSLS's.
Preprints (publication date not yet finalized)
| Hu M, Vink M, Kim C, Derr K, Koss J, D'Amico K, Cheng A, Pulokas J, Ubarretxena-Belandia I, Stokes D. J Struct Biol. 2010 Feb 27. [Epub ahead of print] "Automated Electron Microscopy for Evaluating Two-dimensional Crystallization of Membrane Proteins." |  |
Membrane proteins fulfill many important roles in the cell and represent the target for a large number of therapeutic drugs. Although structure determination of membrane proteins has become a major priority, it has proven to be technically challenging. Electron microscopy of two-dimensional (2D) crystals has the advantage of visualizing membrane proteins in their natural lipidic environment, but has been underutilized in recent structural genomics efforts. To improve the general applicability of electron crystallography, high-throughput methods are needed for screening large numbers of conditions for 2D crystallization, thereby increasing the chances of obtaining well ordered crystals and thus achieving atomic resolution. Previous reports describe devices for growing 2D crystals on a 96-well format. The current report describes a system for automated imaging of these screens with an electron microscope. Samples are inserted with a two-part robot: a SCARA robot for loading samples into the microscope holder, and a Cartesian robot for placing the holder into the electron microscope. A standard JEOL 1230 electron microscope was used, though a new tip was designed for the holder and a toggle switch controlling the airlock was rewired to allow robot control. A computer program for controlling the robots was integrated with the Leginon program, which provides a module for automated imaging of individual samples. The resulting images are uploaded into the Sesame laboratory information management system database where they are associated with other data relevant to the crystallization screen.
| Matthew P. Nicholas, Ertan Eryilmaz, Fabien Ferrage, David Cowburn, and Ranajeet Ghose "Nuclear Spin Resonance Relaxation in Isotropic and Anisotropic Media" Prog. NMR Spectroscopy in press 153 ms pages | 
P |
In this paper, we shall focus primarily on the chemical shift anisotropy (CSA) and dipole-dipole (DD) interactions,
which are by far the two most significant interactions contributing to the relaxation of spin-1/2 (I = 1/2) nuclei
in the solution state. Both are essentially intramolecular interactions modulated by random molecular tumbling in
solution. Since molecular rotational diffusion and internal dynamics determine the nature of the stochastic processes that
modulate the interactions contributing to relaxation, an understanding of the Brownian motion of spin-containing
molecules enables one to determine information regarding their diffusive, shape dependent, and dynamical properties
based upon observed relaxation rates. This determination is the ultimate goal of the theory described in this review.
| Andrew B Waight, James Love, Da-Neng Wang "Structure and mechanism of a pentameric formate channel" Nature Structural & Molecular Biology Published online: 13 December 2009 doi:10.1038/nsmb.1740 |  P |
Formate transport across the inner membrane is a critical step in anaerobic bacterial respiration. Members of the formate/nitrite transport protein family function to shuttle substrate across the cytoplasmic membrane. In bacterial pathogens, the nitrite transport protein is involved in protecting bacteria from peroxynitrite released by host macrophages. We have determined the 2.13- structure of the formate channel FocA from Vibrio cholerae, which reveals a pentamer in which each monomer possesses its own substrate translocation pore. Unexpectedly, the fold of the FocA monomer resembles that found in water and glycerol channels. The selectivity filter in FocA consists of a cytoplasmic slit and a central constriction ring. A 2.5- high-formate structure shows two formate ions bound to the cytoplasmic slit via both hydrogen bonding and van der Waals interactions, providing a structural basis for the substrate selectivity of the channel.
| Shibani Bhattacharya, Zhongping Dai, Jianquan Li, Sabine Baxter, David J.E. Callaway, David Cowburn and Zimei Bu A conformational switch in the sodium/ hydrogen exchange regulatory factor (NHERF) controls autoinhibition and complex formation J Biol Chem in press |
Fox Chase CC |
The mammalian Na+/H+ exchange regulatory factor (NHERF1) is a multi-domain scaffolding protein essential for the regulation of intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, 2 domains that interact with the cytoplasmic
domains of membrane proteins, and a carboxyterminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intra-molecular interactions between PDZ2 and the CT domain, which also includes a PDZ-binding motif (-SNL) at its terminus. We have determined the structures of the isolated and tandem PDZ2CT domains using high-resolution NMR with small angle X-ray scattering data. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a
novel helix-turn-helix subdomain that is allosterically coupled to the canonical PDZ2 domain by a network of hydrophobic interactions. The α helical structural extension tunes the stability of the isolated NHERF1 PDZ2 domain and provides higher affinity to targets. Using small angle neutron
scattering data as constraints for NMR structure refinement, we further demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon forming a complex with ezrin. Based on the structural information we show human disease-causing mutations in PDZ2, R153Q and E225K significantly reduced protein stability in vitro. The potential loss of NHERF1 expressed in cells and failure to assemble membrane complexes is detrimental for normal physiological functions.
| Levin, E. J., M. Quick and M. Zhou (2009). "Crystal structure of a bacterial homologue of the kidney urea transporter. " Nature advance online publication. |  P |
Urea is highly concentrated in the mammalian kidney to produce the osmotic gradient necessary for water re-absorption. Free diffusion of urea across cell membranes is slow owing to its high polarity, and specialized urea transporters have evolved to achieve rapid and selective urea permeation. Here we present the 2.3 Å structure of a functional urea transporter from the bacterium Desulfovibrio vulgaris. The transporter is a homotrimer, and each subunit contains a continuous membrane-spanning pore formed by the two homologous halves of the protein. The pore contains a constricted selectivity filter that can accommodate several dehydrated urea molecules in single file. Backbone and side-chain oxygen atoms provide continuous coordination of urea as it progresses through the filter, and well-placed a-helix dipoles provide further compensation for dehydration energy. These results establish that the urea transporter operates by a channel-like mechanism and reveal the physical and chemical basis of urea selectivity.
| Xu, Y., J. Lorieau and A. E. McDermott "Triosephosphate Isomerase: 15N and 13C Chemical Shift Assignments and Conformational Change upon Ligand Binding by Magic-Angle Spinning Solid-State NMR Spectroscopy." Journal of Molecular Biology In Press, | |
Microcrystalline uniformly 13C,15N -enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.
Articles published in the last few months, or not previously reported
| Piserchio, A., P. A. Nair, S. Shuman and R. Ghose (2010). "Solution NMR Studies of Chlorella Virus DNA Ligase-adenylate." Journal of Molecular Biology 395(2): 291-308. |  |
DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3'-OH/5'-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone N-15 spin relaxation and N-15,H-1 residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5'-phosphate. (C) 2009 Elsevier Ltd. All rights reserved.
| Kuryavyi V, Patel DJ. Solution Structure of a Unique G-Quadruplex Scaffold Adopted by a Guanosine-Rich Human Intronic Sequence. Structure. 2010 Jan 13;18(1):73-82. | |
We report on the solution structure of an unprecedented intramolecular G-quadruplex formed by the guanosine-rich human chl1 intronic d(G(3)-N-G(4)-N(2)-G(4)-N-G(3)-N) 19-mer sequence in K(+)-containing solution. This G-quadruplex, composed of three stacked G-tetrads containing four syn guanines, represents a new folding topology with two unique conformational features. The first guanosine is positioned within the central G-tetrad, in contrast to all previous structures of unimolecular G-quadruplexes, where the first guanosine is part of an outermost G-tetrad. In addition, a V-shaped loop, spanning three G-tetrad planes, contains no bridging nucleotides. The G-quadruplex scaffold is stabilized by a T*G*A triple stacked over the G-tetrad at one end and an unpaired guanosine stacked over the G-tetrad at the other end. Finally, the chl1 intronic DNA G-quadruplex scaffold contains a guanosine base intercalated between an extended G-G step, a feature observed in common with the catalytic site of group I introns. This unique structural scaffold provides a highly specific platform for the future design of ligands specifically targeted to intronic G-quadruplex platforms.
| Abigail S. Haka, Inna Grosheva, Ethan Chiang, Adina R. Buxbaum,Barbara A. Baird, Lynda M. Pierini, and Frederick R. Maxfield " Macrophages Create an Acidic Extracellular Hydrolytic Compartment to Digest Aggregated Lipoproteins " Molecular Biology of the Cell Vol. 20, 4932 4940, December 1, 2009 |  |
A critical event in atherogenesis is the interaction of macrophages with subendothelial lipoproteins. Although most
studies model this interaction by incubating macrophages with monomeric lipoproteins, macrophages in vivo encounter
lipoproteins that are aggregated. The physical features of the lipoproteins require distinctive mechanisms for their uptake.
We show that macrophages create an extracellular, acidic, hydrolytic compartment to carry out digestion of aggregated
low-density lipoproteins. We demonstrate delivery of lysosomal contents to these specialized compartments and their
acidification by vacuolar ATPase, enabling aggregate catabolism by lysosomal acid hydrolases. We observe transient sealing
of portions of the compartments, allowing formation of an extracellular proton gradient. An increase in free cholesterol is
observed in aggregates contained in these compartments. Thus, cholesteryl ester hydrolysis can occur extracellularly in a
specialized compartment, a lysosomal synapse, during the interaction of macrophages with aggregated low-density lipoprotein.
A detailed understanding of these processes is essential for developing strategies to prevent atherosclerosis.
| Wu, H. Y., S. C. Shekar, R. J. Flinn, M. El-Sibai, B. S. Jaiswal, K. I. Sen, V. Janakiraman, S. Seshagiri, G. J. Gerfen, M. E. Girvin and J. M. Backer (2009). "Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110 α and are disrupted in oncogenic p85 mutants." Proceedings Of The National Academy Of Sciences Of The United States Of America 106(48): 20258-20263. |  P |
We previously proposed a model of Class IA PI3K regulation in which p85 inhibition of p110 α requires (i) an inhibitory contact between the p85 nSH2 domain and the p110 α helical domain, and (ii) a contact between the p85 nSH2 and iSH2 domains that orients the nSH2 so as to inhibit p110 α. We proposed that oncogenic truncations of p85 fail to inhibit p110 due to a loss of the iSH2-nSH2 contact. However, we now find that within the context of a minimal regulatory fragment of p85 (the nSH2-iSH2 fragment, termed p85ni), the nSH2 domain rotates much more freely (tau(C) approximate to 12.7 ns) than it could if it were interacting rigidly with the iSH2 domain. These data are not compatible with our previous model. We therefore tested an alternative model in which oncogenic p85 truncations destabilize an interface between the p110 α C2 domain (residue N345) and the p85 iSH2 domain (residues D560 and N564). p85ni-D560K/N564K shows reduced inhibition of p110 α, similar to the truncated p85ni-572(STOP). Conversely, wild-type p85ni poorly inhibits p110 α N345K. Strikingly, the p110 α N345K mutant is inhibited to the same extent by the wild-type or truncated p85ni, suggesting that mutation of p110 α-N345 is not additive with the p85ni-572(STOP) mutation. Similarly, the D560K/N564K mutation is not additive with the p85ni-572(STOP) mutant for downstream signaling or cellular transformation. Thus, our data suggests that mutations at the C2-iSH2 domain contact and truncations of the iSH2 domain, which are found in human tumors, both act by disrupting the C2-iSH2 domain interface.
| Rakus, J. F., C. Kalyanaraman, A. A. Fedorov, E. V. Fedorov, F. P. Mills-Groninger, R. Toro, J. Bonanno, K. Bain, J. M. Sauder, S. K. Burley, S. C. Almo, M. P. Jacobson and J. A. Gerlt (2009). "Computation-facilitated assignment of the function in the enolase superfamily: a regiochemically distinct galactarate dehydratase from Oceanobacillus iheyensis." Biochemistry 48(48): 11546-58. | |
The structure of an uncharacterized member of the enolase superfamily from Oceanobacillus iheyensis (GI 23100298, IMG locus tag Ob2843, PDB entry 2OQY ) was determined by the New York SGX Research Center for Structural Genomics (NYSGXRC). The structure contained two Mg(2+) ions located 10.4 A from one another, with one located in the canonical position in the (beta/alpha)(7)beta-barrel domain (although the ligand at the end of the fifth beta-strand is His, unprecedented in structurally characterized members of the superfamily); the second is located in a novel site within the capping domain. In silico docking of a library of mono- and diacid sugars to the active site predicted a diacid sugar as a likely substrate. Activity screening of a physical library of acid sugars identified galactarate as the substrate (k(cat) = 6.8 s(-1), K(M) = 620 microM, k(cat)/K(M) = 1.1 x 10(4) M(-1) s(-1)), allowing functional assignment of Ob2843 as galactarate dehydratase (GalrD-II). The structure of a complex of the catalytically impaired Y90F mutant with Mg(2+) and galactarate allowed identification of a Tyr 164-Arg 162 dyad as the base that initiates the reaction by abstraction of the alpha-proton and Tyr 90 as the acid that facilitates departure of the beta-OH leaving group. The enzyme product is 2-keto-d-threo-4,5-dihydroxyadipate, the enantiomer of the product obtained in the GalrD reaction catalyzed by a previously characterized bifunctional l-talarate/galactarate dehydratase (TalrD/GalrD). On the basis of the different active site structures and different regiochemistries, we recognize that these functions represent an example of apparent, not actual, convergent evolution of function. The structure of GalrD-II and its active site architecture allow identification of the seventh functionally and structurally characterized subgroup in the enolase superfamily. This study provides an additional example in which an integrated sequence- and structure-based strategy employing computational approaches is a viable approach for directing functional assignment of unknown enzymes discovered in genome projects.
| Burke, V., C. Williams, M. Sukumaran, S. S. Kim, H. Li, X. H. Wang, M. K. Gorny, S. Zolla-Pazner and X. P. Kong (2009). "Structural basis of the cross-reactivity of genetically related human anti-HIV-1 mAbs: implications for design of V3-based immunogens." Structure 17(11): 1538-46. | |
Human monoclonal antibodies 447-52D and 537-10D, both coded by the VH3 gene and specific for the third variable region (V3) of the HIV-1 gp120, were found to share antigen-binding structural elements including an elongated CDR H3 forming main-chain interactions with the N terminus of the V3 crown. However, water-mediated hydrogen bonds and a unique cation-pi sandwich stacking allow 447-52D to be broadly reactive with V3 containing both the GPGR and GPGQ crown motifs, while the deeper binding pocket and a buried Glu in the binding site of 537-10D limit its reactivity to only V3 containing the GPGR motif. Our results suggest that the design of immunogens for anti-V3 antibodies should avoid the Arg at the V3 crown, as GPGR-containing epitopes appear to select for B cells making antibodies of narrower specificity than V3 that carry Gln at this position.
| Q Yin, B Lamothe, B Darnay, H Wu, Structural Basis for the Lack of E2 Interaction in the RING Domain of TRAF2, Biochemistry, 48, 10558 10567 (2009). | |
TRAF proteins are intracellular signal transducers for a number of immune receptor superfamilies. Specifically, TRAF2 interacts with members of the TNF receptor superfamily and connects the receptors to downstream signaling proteins. It has been assumed that TRAF2 is a ubiquitin ligase like TRAF6 and mediates K63-linked polyubiquitination of RIP1, a kinase pivotal in TNFalpha-induced NF-kappaB activation. Here we report the crystal structure of the RING and the first zinc finger domains of TRAF2. We show that the TRAF2 RING structure is very different from the known TRAF6 RING structure. The differences are multifaceted, including amino acid differences at the critical Ubc13-interacting site, local conformational differences, and a unique nine-residue insertion between the RING domain and the first zinc finger in TRAF2. These structural differences prevent TRAF2 from interacting with Ubc13 and other related E2s via steric clash and unfavorable interfaces. Our structural observation should prompt a re-evaluation of the role of TRAF2 in TNFalpha signaling and may indicate that TRAF2-associated proteins such as cIAPs may be the ubiquitin ligases for NF-kappaB signaling.
| Z Liu, Y Gosser, P Baker, Y Ravee, H Li, G Butterfoss, X Kong, R Gross, J Montclare, Structural and Functional Studies of Aspergillus oryzae Cutinase: Enhanced Thermostability and Hydrolytic Activity of Synthetic Ester and Polyester Degradation, J. Am. Chem. Soc., 131, 15711 15716 (2009). |  P |
Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically favored catalytic triad with a continuous and deep groove. These structural features of A. oryzae cutinase are proposed to result in an improved hydrolytic activity and altered substrate specificity profile, enhanced thermostability, and remarkable reactivity toward the degradation of the synthetic polyester polycaprolactone. The results presented here provide insight into engineering new cutinase-inspired biocatalysts with tailor-made properties.
| Stiegler, A. L., S. J. Burden and S. R. Hubbard (2009). "Crystal structure of the frizzled-like cysteine-rich domain of the receptor tyrosine kinase MuSK." J Mol Biol 393(1): 1-9. | |
Muscle-specific kinase (
MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of
MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of
MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the
MuSK Fz-CRD at 2.1 A resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of
MuSK, rapsyn, and acetylcholine receptors at the NMJ.
| J Cheung, M Le-Khac, W Hendrickson, Crystal Structure of a Histidine Kinase Sensor Domain with Similarity to Periplasmic Binding Proteins, Proteins: Struc. Func. Bioinformatics, Oct 77, 235-241 (2009). | |
Histidine kinase receptors are elements of the twocomponent
signal transduction systems commonly found
in bacteria and lower eukaryotes, where they are crucial
for environmental adaptation through the coupling of
extracellular changes to intracellular responses.1,2 The
typical two-component system consists of a membranespanning
histidine kinase sensor and a cytoplasmic
response regulator. In the classic system, extracellular signals
such as small molecule ligands and ions are detected
by the periplasmic sensor domain of the histidine kinase
receptor, which modulates the catalytic activity of the
cytoplasmic histidine kinase domain and promotes ATPdependent
autophosphorylation of a conserved histidine
residue. The phosphate is subsequently transferred to a
conserved aspartate of the cognate response regulator
through a phospho-transfer mechanism, and the activity
of the response regulator is modulated in turn.3,4 The
response regulator often controls gene transcription or
flagellar rotation, and thus allows cellular adaptation to
an environmental signal.
| Punta, M., Love, J., Handelman, S., Hunt, J., Shapiro, L., Hendrickson, W., and Rost, B. Structural genomics target selection for the New York consortium on membrane protein structure. Journal of Structural and Functional Genomics. in press. | |
The New York Consortium on Membrane Protein Structure (NYCOMPS), a part of the Protein Structure Initiative (PSI) in the USA, has as its mission to establish a high-throughput pipeline for determination of novel integral membrane protein structures. Here we describe our current target selection protocol, which applies structural genomics approaches informed by the collective experience of our team of investigators. We first extract all annotated proteins from our reagent genomes, i.e. the 96 fully sequenced prokaryotic genomes from which we clone DNA. We filter this initial pool of sequences and obtain a list of valid targets. NYCOMPS defines valid targets as those that, among other features, have at least two predicted transmembrane helices, no predicted long disordered regions and, except for community nominated targets, no significant sequence similarity in the predicted transmembrane region to any known protein structure. Proteins that feed our experimental pipeline are selected by defining a protein seed and searching the set of all valid targets for proteins that are likely to have a transmembrane region structurally similar to that of the seed. We require sequence similarity aligning at least half of the predicted transmembrane region of seed and target. Seeds are selected according to their feasibility and/or biological interest, and they include both centrally selected targets and community nominated targets. As of December 2008, over 6,000 targets have been selected and are currently being processed by the experimental pipeline. We discuss how our target list may impact structural coverage of the membrane protein space.
| O'Connell NE, Grey MJ, Tang Y, Kosuri P, Miloushev VZ, Raleigh DP, Palmer AG 'Partially folded equilibrium intermediate of the villin headpiece HP67 defined by 13C relaxation dispersion.' J Biomol NMR 2009 45(1-2):85-98 | |
Identification and characterization of ensembles of intermediate states remains an important objective in describing protein folding in atomic detail. The 67-residue villin headpiece, HP67, consists of an N-terminal subdomain (residues 10 42) that transiently unfolds at equilibrium under native-like conditions and a highly stable C-terminal subdomain (residues 43 76). The transition between folded and unfolded states of the N-terminal domain has been characterized previously by 15N NMR relaxation dispersion measurements (Grey et al. in J Mol Biol 355:1078, 2006). In the present work, 13C spin relaxation was used to further characterize backbone and hydrophobic core contributions to the unfolding process. Relaxation of 13Cα spins was measured using the Hahn echo technique at five static magnetic fields (11.7, 14.1, 16.4, 18.8, and 21.1 T) and the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion method at a static magnetic field of 14.1 T. Relaxation of methyl 13C spins was measured using CPMG relaxation dispersion experiments at static magnetic fields of 14.1 and 18.8 T. Results for 13C and 15N spins yielded a consistent model in which the partially unfolded intermediate state of the N-terminal subdomain maintains residual structure for residues near the unprotonated His41 imidazole ring and in the interface between the N- and C-terminal subdomains. In addition, a second faster process was detected that appears to represent local dynamics within the folded state of the molecule and is largely confined to the hydrophobic interface between the N- and C-terminal subdomains.
| Mohideen, F., Capili, A.D., Bilimoria, P.M., Yamada, T., Bonni, A., and Lima, C.D. A molecular basis for phosphorylation-dependent SUMO conjugation by the E2 Ubc9 (2009) Nat Struct. Mol. Bio. 16(9):945-52. | P |
Phosphorylation and small ubiquitin-like modifier (SUMO) conjugation contribute to the spatial and temporal regulation of substrates containing phosphorylation-dependent SUMO consensus motifs (PDSMs). Myocyte-enhancement factor 2 (MEF2) is a transcription factor and PDSM substrate whose modification by SUMO drives postsynaptic dendritic differentiation. NMR analysis revealed that the human SUMO E2 interacted with model substrates for phosphorylated and nonphosphorylated MEF2 in similar extended conformations. Mutational and biochemical analysis identified a basic E2 surface that enhanced SUMO conjugation to phosphorylated PDSM substrates MEF2 and heat-shock transcription factor 1 (HSF1), but not to nonphosphorylated MEF2 or HSF1, nor the non-PDSM substrate p53. Mutant ubiquitin-conjugating enzyme UBC9 isoforms defective in promoting SUMO conjugation to phosphorylated MEF2 in vitro and in vivo also impair postsynaptic differentiation in organotypic cerebellar slices. These data support an E2-dependent mechanism that underlies phosphorylation-dependent SUMO conjugation in pathways that range from the heat-shock response to nuclear hormone signaling to brain development.
| Martinez-Hackert, E. and W. A. Hendrickson (2009). "Promiscuous substrate recognition in folding and assembly activities of the trigger factor chaperone." Cell 138(5): 923-34. | P |
Trigger factor (TF) is a molecular chaperone that binds to bacterial ribosomes where it contacts emerging nascent chains, but TF is also abundant free in the cytosol where its activity is less well characterized. In vitro studies show that TF promotes protein refolding. We find here that ribosome-free TF stably associates with and rescues from misfolding a large repertoire of full-length proteins. We identify over 170 members of this cytosolic Escherichia coli TF substrate proteome, including ribosomal protein S7. We analyzed the biochemical properties of a TF:S7 complex from Thermotoga maritima and determined its crystal structure. Thereby, we obtained an atomic-level picture of a promiscuous chaperone in complex with a physiological substrate protein. The structure of the complex reveals the molecular basis of substrate recognition by TF, indicates how TF could accelerate protein folding, and suggests a role for TF in the biogenesis of protein complexes.
| Moore, J. O. and W. A. Hendrickson (2009). "Structural Analysis of Sensor Domains from the TMAO-Responsive Histidine Kinase Receptor TorS." Structure 17(9): 1195-1204. | |
Histidine kinase receptors respond to diverse signals and mediate signal transduction across the plasma membrane in all prokaryotes and certain eukaryotes. Each receptor is part of a two-component system that regulates a particular cellular process. Organisms that use trimethylamine-N-oxide (TMAO) as a terminal electron acceptor typically control their anaerobic respiration through the TMAO reductase (Tor) pathway, which the TorS histidine kinase activates when sensing TMAO in the environment. We have determined crystal structures for the periplasmic sensor domains of TorS receptors from Escherichia coli and Vibrio parahaemolyticus. TorS sensor domains have a novel fold consisting of a membrane-proximal right-handed four-helical bundle and a membrane-distal left-handed four-helical bundle, but conformational dispositions differ significantly in the two structures. Isolated TorS sensor domains dimerize in solution; and from comparisons with dimeric NarX and Tar sensors, we postulate that signaling through TorS dimers involves a piston-type displacement between helices.
| Wei H, Cheng RH, Berriman J, Rice WJ, Stokes DL, Katz A, Morgan DG, Gottlieb P 'Three-dimensional structure of the enveloped bacteriophage φ12: an incomplete T = 13 lattice is superposed on an enclosed T = 1 shell.' PLoS One 2009;4(9) |
|
Bacteriophage φ12 is a member of the Cystoviridae, a unique group of lipid containing membrane enveloped bacteriophages that infect the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola. The genomes of the virus species contain three double-stranded (dsRNA) segments, and the virus capsid itself is organized in multiple protein shells. The segmented dsRNA genome, the multi-layered arrangement of the capsid and the overall viral replication scheme make the Cystoviridae similar to the Reoviridae. We present structural studies of cystovirus phi12 obtained using cryo-electron microscopy and image processing techniques. We have collected images of isolated phi12 virions and generated reconstructions of both the entire particles and the polymerase complex (PC). We find that in the nucleocapsid (NC), the phi12 P8 protein is organized on an incomplete T = 13 icosahedral lattice where the symmetry axes of the T = 13 layer and the enclosed T = 1 layer of the PC superpose. This is the same general protein-component organization found in phi6 NC's but the detailed structure of the entire phi12 P8 layer is distinct from that found in the best classified cystovirus species phi6. In the reconstruction of the NC, the P8 layer includes protein density surrounding the hexamers of P4 that sit at the 5-fold vertices of the icosahedral lattice. We believe these novel features correspond to dimers of protein P7. In conclusion, we have determined that the φ12 NC surface is composed of an incomplete T = 13 P8 layer forming a net-like configuration. The significance of this finding in regard to cystovirus assembly is that vacancies in the lattice could have the potential to accommodate additional viral proteins that are required for RNA packaging and synthesis.
| Nguyen, T. T., A. A. Fedorov, L. Williams, E. V. Fedorov, Y. Li, C. Xu, S. C. Almo and F. M. Raushel (2009). "The mechanism of the reaction catalyzed by uronate isomerase illustrates how an isomerase may have evolved from a hydrolase within the amidohydrolase superfamily." Biochemistry 48(37): 8879-90. | |
Uronate isomerase (URI) catalyzes the reversible isomerization of D-glucuronate to D-fructuronate and of D-galacturonate to D-tagaturonate. URI is a member of the amidohydrolase superfamily (AHS), a highly divergent group of enzymes that catalyze primarily hydrolytic reactions. The chemical mechanism and active site structure of URI were investigated in an attempt to improve our understanding of how an active site template that apparently evolved to catalyze hydrolytic reactions has been reforged to catalyze an isomerization reaction. The pH-rate profiles for k(cat) and k(cat)/K(m) for URI from Escherichia coli are bell-shaped and indicate that one group must be unprotonated and another residue must be protonated for catalytic activity. Primary isotope effects on the kinetic constants with [2-2H]-D-glucuronate and the effects of changes in solvent viscosity are consistent with product release being the rate-limiting step. The X-ray structure of Bh0493, a URI from Bacillus halodurans, was determined in the presence of the substrate D-glucuronate. The bound complex showed that the mononuclear metal center in the active site is ligated to the C-6 carboxylate and the C-5 hydroxyl group of the substrate. This hydroxyl group is also hydrogen bonded to Asp-355 in the same orientation as the hydroxide or water is bound in those members of the AHS that catalyze hydrolytic reactions. In addition, the C-2 and C-3 hydroxyl groups of the substrate are hydrogen bonded to Arg-357 and the carbonyl group at C-1 is hydrogen bonded to Tyr-50. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 from the end of beta-strand 8 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the proposed cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-355.
| Yu B, Hunt JF. Proc Natl Acad Sci U S A. 2009 Aug 25;106(34):14315-20. Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB. | P |
Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N(6)-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher k(cat) and K(m) for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this "k(cat)/K(m) compensation," which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O(2) substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O(2) binding before nucleotide substrate binding.
| Liu D, Xu R, Cowburn D 'Segmental isotopic labeling of proteins for nuclear magnetic resonance.' Methods Enzymol 2009; 462 :151-75 | |
Nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology. It is not only a powerful method for elucidating the three-dimensional structures under near physiologicalconditions but also a convenient method for studying protein-ligand interactions and protein dynamics. A major drawback of macromolecular NMR is its size limitation, caused by slower tumbling rates and greater complexity of the spectra as size increases. Segmental isotopic labeling allows for specific segment(s) within a protein to be selectively examined by NMR, thus significantly reducing the spectral complexity for large proteins and allowing for the application of a variety of solution-based NMR strategies. Two related approaches are generally used in the segmental isotopic labeling of proteins: expressed protein ligation and protein trans-splicing. Here, we describe the methodology and recent application of expressed protein ligation and protein trans-splicing for NMR structural studies of proteins and protein complexes. We also describe the protocol used in our lab for the segmental isotopic labeling of a 50-kDa
protein Csk (C-terminal Src kinase) using expressed protein ligation methods.
| Depetris RS, Wu J, Hubbard SR 'Structural and functional studies of the Ras-associating and pleckstrin-homology domains of Grb10 and Grb14.' Nat Struct Mol Biol 2009 Aug; 16 (8):833-9 |
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Growth factor receptor binding proteins Grb7, Grb10 and Grb14 are adaptor proteins containing a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region and a C-terminal Src-homology-2 domain. Previous structural studies showed that the Grb14 BPS region binds as a pseudosubstrate inhibitor in the tyrosine kinase domain of the insulin receptor to suppress insulin signaling. Here we report the crystal structure of the RA and PH domains of Grb10 at 2.6-Å resolution. The structure reveals that these two domains, along with the intervening linker, form an integrated, dimeric structural unit. Biochemical studies demonstrated that Grb14 binds to activated Ras, which may serve as a timing mechanism for downregulation of insulin signaling. Our results illuminate the membrane-recruitment mechanisms not only of Grb7, Grb10 and Grb14 but also of MIG-10, Rap1-interacting adaptor molecule, lamellipodin and Pico, proteins involved in actin-cytoskeleton rearrangement that share a structurally related RA-PH tandem unit.
| A Sakai, A Fedorov, E Fedorov, A Schnoes, M Glasner, S Burley, P Babbitt, S Almo, J Gerlt, Evolution of Enzymatic Activities in the Enolase Superfamily: Stereochemically Distinct Mechanisms in Two Families of cis,cis-Muconate Lactonizing Enzymes, Biochemistry, 48, 1445 1453 (2009). | |
The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k(cat)/K(m) = 5.8 x 10(6) M(-1) s(-1)), N-acetyl-d-glutamate (k(cat)/K(m) = 5.2 x 10(6) M(-1) s(-1)), and l-methionine-d-glutamate (k(cat)/K(m) = 3.4 x 10(5) M(-1) s(-1)). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k(cat)/K(m) = 3.2 x 10(4) M(-1) s(-1)), N-acetyl-d-tryptophan (k(cat)/K(m) = 4.1 x 10(4) M(-1) s(-1)), and l-tyrosine-d-leucine (k(cat)/K(m) = 1.5 x 10(4) M(-1) s(-1)). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the alpha-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional approximately 250 sequences identified as members of this group suggest that there are no simple motifs that allow prediction of substrate specificity for most of these unknowns, highlighting the challenges for computational annotation of some groups of homologous proteins.
| J Kalinina, S Byron, H Makarenkova, S Olsen, A Eliseenkova, W Larochelle, M Dhanabal, S Blais, M Mohammadi, Homodimerization Controls the Fibroblast Growth Factor 9 Subfamily's Receptor Binding and Heparan Sulfate-Dependent Diffusion in the Extracellular Matrix, Mol. Cell. Bio., 29, 4663-4678 (2009). | |
Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.
| Pinar Akcora and Sanat K. Kumar "ANISOTROPIC SELF-ASSEMBLY OF NANOPARTICLES IN COMPOSITES" WO 2009078985 20090625 | |
The subject matter disclosed and claimed herein is directed to anisotropic nanoparticulate structures, to methods for their preparation, and to methods of use. In various embodiments, the disclosed subject matter provides an anisotropic self-assembled structure comprising a plurality of substantially isotropic grafted nanoparticles in a polymeric matrix, the self-assembled structure comprising (a) a one-dimensional string of the grafted nanoparticles within the matrix, the string having a width of one nanoparticle and a length of 1-10 nanoparticles, (b) a substantially two-dimensional sheet of the grafted nanoparticles within the matrix, the sheet having a length and a breadth of at least one micron respectively, wherein the sheet is about 2-5 nanoparticles in thickness, or (c) a plurality of three dimensionally interconnected structures where the width of each of the structures is 1-10 nanoparticles; wherein each grafted nanoparticle comprises a substantially spherical inorganic core nanoparticle and an organic brush structure grafted thereto, wherein the brush structure comprises a plurality of grafted polymeric chains of a weight average molecular weight Mg, the polymeric matrix has a weight average molecular weight of M, and there is an average number of grafted polymeric chains per nm2 of surface of the inorganic core nanoparticles D. ... We have conducted TEM tomography using Tecnai F20 (20OkV FEG) electron microscope at the New York Structural Biology Center. ...
| Yu LP, Xiang S, Lasso G, Gil D, Valle M, Tong L 'A symmetrical tetramer for S. aureus pyruvate carboxylase in complex with coenzyme A.' Structure. 2009 Jun 10; 17 (6):823-32 | |
Pyruvate carboxylase (PC) is a conserved metabolic enzyme with important cellular functions. We report crystallographic and cryo-electron microscopy (EM) studies of Staphylococcus aureus PC (SaPC) in complex with acetyl-CoA, an allosteric activator, and mutagenesis, biochemical, and structural studies of the biotin binding site of its carboxyltransferase (CT) domain. The disease-causing A610T mutation abolishes catalytic activity by blocking biotin binding to the CT active site, and Thr908 might play a catalytic role in the CT reaction. The crystal structure of SaPC in complex with CoA reveals a symmetrical nature of the tetramer. These observations are in sharp contrast to the highly asymmetrical teramer of Rhizobium etli PC in complex with ethyl-CoA. Our structural information suggests that acetyl-CoA promotes a conformation for the dimer of the biotin carboxylase domain of PC that might be catalytically more competent.
| Libich, David S.; Schwalbe, Martin; Kate, Sachin; Venugopal, Hariprasad; Claridge, Jolyon K.; Edwards, Patrick J. B.; Dutta, Kaushik; Pascal, Steven M. Intrinsic disorder and coiled-coil formation in prostate apoptosis response factor 4 FEBS Journal, Volume 276, Number 14, July 2009 , pp. 3710-3728 | Massey U.
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Prostate apoptosis response factor-4 (Par-4) is an ubiquitously expressed pro-apoptotic and tumour suppressive protein that can both activate cell-death mechanisms and inhibit pro-survival factors. Par-4 contains a highly conserved coiled-coil region that serves as the primary recognition domain for a large number of binding partners. Par-4 is also tightly regulated by the aforementioned binding partners and by post-translational modifications. Biophysical data obtained in the present study indicate that Par-4 primarily comprises an intrinsically disordered protein. Bioinformatic analysis of the highly conserved Par-4 reveals low sequence complexity and enrichment in polar and charged amino acids. The high proteolytic susceptibility and an increased hydrodynamic radius are consistent with a largely extended structure in solution. Spectroscopic measurements using CD and NMR also reveal characteristic features of intrinsic disorder. Under physiological conditions, the data obtained show that Par-4 self-associates via the C-terminal domain, forming a coiled-coil. Interruption of self-association by urea also resulted in loss of secondary structure. These results are consistent with the stabilization of the coiled-coil motif through an intramolecular association.
| Yin, Q., S. C. Lin, B. Lamothe, M. Lu, Y. C. Lo, G. Hura, L. Zheng, R. L. Rich, A. D. Campos, D. G. Myszka, M. J. Lenardo, B. G. Darnay and H. Wu (2009). "E2 interaction and dimerization in the crystal structure of TRAF6." Nat Struct Mol Biol 16(6): 658-66. | P |
Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-kappaB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors.
| Chan, K. K., B. M. Wood, A. A. Fedorov, E. V. Fedorov, H. J. Imker, T. L. Amyes, J. P. Richard, S. C. Almo and J. A. Gerlt (2009). "Mechanism of the orotidine 5'-monophosphate decarboxylase-catalyzed reaction: evidence for substrate destabilization." Biochemistry 48(24): 5518-31. | |
The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) involves a stabilized anionic intermediate, although the structural basis for the rate acceleration (k(cat)/k(non), 7.1 x 10(16)) and proficiency [(k(cat)/K(M))/k(non), 4.8 x 10(22) M(-1)] is uncertain. That the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC) catalyze the exchange of H6 of the UMP product with solvent deuterium allows an estimate of a lower limit on the rate acceleration associated with stabilization of the intermediate and its flanking transition states (>or=10(10)). The origin of the "missing" contribution, <or=10(7) ( approximately 10(17) total - >or=10(10)), is of interest. Based on structures of liganded complexes, unfavorable electrostatic interactions between the substrate carboxylate group and a proximal Asp (Asp 70 in MtOMPDC and Asp 91 in ScOMPDC) have been proposed to contribute to the catalytic efficiency [Wu, N., Mo, Y., Gao, J., and Pai, E. F. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 2017-2022]. We investigated that hypothesis by structural and functional characterization of the D70N and D70G mutants of MtOMPDC and the D91N mutant of ScOMPDC. The substitutions for Asp 70 in MtOMPDC significantly decrease the value of k(cat) for decarboxylation of FOMP (a more reactive substrate analogue) but have little effect on the value of k(ex) for exchange of H6 of FUMP with solvent deuterium; the structures of wild-type MtOMPDC and its mutants are superimposable when complexed with 6-azaUMP. In contrast, the D91N mutant of ScOMPDC does not catalyze exchange of H6 of FUMP; the structures of wild-type ScOMPDC and its D91N mutant are not superimposable when complexed with 6-azaUMP, with differences in both the conformation of the active site loop and the orientation of the ligand vis a vis the active site residues. We propose that the differential effects of substitutions for Asp 70 of MtOMPDC on decarboxylation and exchange provide additional evidence for a carbanionic intermediate as well as the involvement of Asp 70 in substrate destabilization.
| G Liu, F Forouhar, A Eletsky, H Atreya, J Aramini, R Xiao, G Montelione, J Hunt, T Szyperski, NMR and X-RAY Structures of Human E2-like Ubiquitin-fold Modifier Conjugating Enzyme 1 (UFC1) Reveal Structural and Functional Conservation in the Metazoan UFM1-UBA5-UFC1 Ubiquination Pathway, J. Struct. Funct. Genomics, 10, 127-136 (2009) | |
For cell regulation, E2-like ubiquitin-fold modifier conjugating enzyme 1 (Ufc1) is involved in the transfer of ubiquitin-fold modifier 1 (Ufm1), a ubiquitin like protein which is activated by E1-like enzyme Uba5, to various target proteins. Thereby, Ufc1 participates in the very recently discovered Ufm1-Uba5-Ufc1 ubiquination pathway which is found in metazoan organisms. The structure of human Ufc1 was solved by using both NMR spectroscopy and X-ray crystallography. The complementary insights obtained with the two techniques provided a unique basis for understanding the function of Ufc1 at atomic resolution. The Ufc1 structure consists of the catalytic core domain conserved in all E2-like enzymes and an additional N-terminal helix. The active site Cys(116), which forms a thio-ester bond with Ufm1, is located in a flexible loop that is highly solvent accessible. Based on the Ufc1 and Ufm1 NMR structures, a model could be derived for the Ufc1-Ufm1 complex in which the C-terminal Gly(83) of Ufm1 may well form the expected thio-ester with Cys(116), suggesting that Ufm1-Ufc1 functions as described for other E1-E2-E3 machineries. alpha-helix 1 of Ufc1 adopts different conformations in the crystal and in solution, suggesting that this helix plays a key role to mediate specificity.
| Neumoin A, Cohen LS, Arshava B, Tantry S, Becker JM, Zerbe O, Naider F. 'Structure of a double transmembrane fragment of a G-protein-coupled receptor in micelles.' Biophys J. 2009 Apr 22; 96 (8):3187-96. | |
The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for a-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [15N], [15N 13C], [15N, 13C, 2H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei. The NMR investigation revealed the secondary structure of specific residues of TM1-TM2. TALOS constraints and NOE connectivities were used to calculate a structure for TM1-TM2 that was highlighted by the presence of three a-helices encompassing residues 39 47, 49 72, and 80 103, with higher flexibility around the internal Arg58 site of TM1. RMSD values of individually superimposed helical segments 39 47, 49 72, and 80 103 were 0.25 50.10 Å , 0.405 0.13 Å , and 0.5750.19 Å , respectively. Several long-range interhelical connectivities supported the folding of TM1-TM2 into a tertiary structure typified by a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G56VRSG60 region. 15N-relaxation and hydrogen deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor.
| Acehan D, Khuchua Z, Houtkooper RH, Malhotra A, Kaufman J, Vaz FM, Ren M, Rockman HA, Stokes DL, Schlame M. 'Distinct effects of tafazzin deletion in differentiated and undifferentiated mitochondria.' Mitochondrion 2009 Apr; 9 (2):86-95. | |
Tafazzin is a conserved mitochondrial protein that is required to maintain normal content and composition of cardiolipin. We used electron tomography to investigate the effect of tafazzin deletion on mitochondrial structure and found that cellular differentiation plays a crucial role in the manifestation of abnormalities. This conclusion was reached by comparing differentiated cardiomyocytes with embryonic stem cells from mouse and by comparing different tissues from Drosophila melanogaster. The data suggest that tafazzin deficiency affects cardiolipin in all mitochondria, but significant alterations of the ultrastructure, such as remodeling and aggregation of inner membranes, will only occur after specific differentiation.
| D Xiang, P Kolb, A Fedorov, M Meier, L Fedorov, T Nguyen, R Sterner, S Almo, B Shoichet, F Raushel, Functional Annotation and Three-Dimensional Structure of Dr0930 from Deinococcus radiodurans, a Close Relative of Phosphotriesterase in the Amidohydrolase Superfamily, Biochemistry, 48, 2237 2247 (2009). | |
Dr0930, a member of the amidohydrolase superfamily in Deinococcus radiodurans, was cloned, expressed, and purified to homogeneity. The enzyme crystallized in the space group P3121, and the structure was determined to a resolution of 2.1 A. The protein folds as a (beta/alpha)7beta-barrel, and a binuclear metal center is found at the C-terminal end of the beta-barrel. The purified protein contains a mixture of zinc and iron and is intensely purple at high concentrations. The purple color was determined to be due to a charge transfer complex between iron in the beta-metal position and Tyr-97. Mutation of Tyr-97 to phenylalanine or complexation of the metal center with manganese abolished the absorbance in the visible region of the spectrum. Computational docking was used to predict potential substrates for this previously unannotated protein. The enzyme was found to catalyze the hydrolysis of delta- and gamma-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was delta-nonanoic lactone with a kcat/Km of 1.6 x 10(6) M-1 s-1. Dr0930 was also found to catalyze the very slow hydrolysis of paraoxon with values of kcat and kcat/Km of 0.07 min-1 and 0.8 M-1 s-1, respectively. The amino acid sequence identity to the phosphotriesterase (PTE) from Pseudomonas diminuta is 30%. The eight substrate specificity loops were transplanted from PTE to Dr0930, but no phosphotriesterase activity could be detected in the chimeric PTE-Dr0930 hybrid. Mutation of Phe-26 and Cys-72 in Dr0930 to residues found in the active site of PTE enhanced the kinetic constants for the hydrolysis of paraoxon. The F26G/C72I mutant catalyzed the hydrolysis of paraoxon with a kcat of 1.14 min-1, an increase of 16-fold over the wild-type enzyme. These results support previous proposals that phosphotriesterase activity evolved from an ancestral parent enzyme possessing lactonase activity.
| Xiang, S., A. Cooper-Morgan, X. Jiao, M. Kiledjian, J. L. Manley and L. Tong (2009). "Structure and function of the 5' - - >3' exoribonuclease Rat1 and its activating partner Rai1." Nature 458(7239): 784-8. | P |
The 5'- - >3' exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with crucial functions in RNA metabolism and RNA interference. XRN2, or Rat1 in yeast, functions primarily in the nucleus and also has an important role in transcription termination by RNA polymerase II (refs 7-14). Rat1 exoribonuclease activity is stimulated by the protein Rai1 (refs 15, 16). Here we report the crystal structure at 2.2 A resolution of Schizosaccharomyces pombe Rat1 in complex with Rai1, as well as the structures of Rai1 and its murine homologue Dom3Z alone at 2.0 A resolution. The structures reveal the molecular mechanism for the activation of Rat1 by Rai1 and for the exclusive exoribonuclease activity of Rat1. Biochemical studies confirm these observations, and show that Rai1 allows Rat1 to degrade RNAs with stable secondary structure more effectively. There are large differences in the active site landscape of Rat1 compared to related and PIN (PilT? N terminus) domain-containing nucleases. Unexpectedly, we identified a large pocket in Rai1 and Dom3Z that contains highly conserved residues, including three acidic side chains that coordinate a divalent cation. Mutagenesis and biochemical studies demonstrate that Rai1 possesses pyrophosphohydrolase activity towards 5' triphosphorylated RNA. Such an activity is important for messenger RNA degradation in bacteria, but this is, to our knowledge, the first demonstration of this activity in eukaryotes and suggests that Rai1/Dom3Z may have additional important functions in RNA metabolism.
| Liu, J., Y. Deng, A. K. Dey, J. P. Moore and M. Lu (2009). "Structure of the HIV-1 gp41 membrane-proximal ectodomain region in a putative prefusion conformation." Biochemistry 48(13): 2915-23. | |
The conserved membrane-proximal external region (MPER) of the HIV-1 gp41 envelope protein is the established target for very rare but broadly neutralizing monoclonal antibodies (NAbs) elicited during natural human infection. Nevertheless, attempts to generate an HIV-1 neutralizing antibody response with immunogens bearing MPER epitopes have met with limited success. Here we show that the MPER peptide (residues 662-683) forms a labile alpha-helical trimer in aqueous solution and report the crystal structure of this autonomous folding subdomain stabilized by addition of a C-terminal isoleucine zipper motif. The structure reveals a parallel triple-stranded coiled coil in which the neutralization epitope residues are buried within the interface between the associating MPER helices. Accordingly, both the 2F5 and 4E10 NAbs recognize the isolated MPER peptide but fail to bind the trimeric MPER subdomain. We propose that the trimeric MPER structure represents the prefusion conformation of gp41, preceding the putative prehairpin intermediate and the postfusion trimer-of-hairpins structure. As such, the MPER trimer should inform the design of new HIV-1 immunogens to elicit broadly neutralizing antibodies.
| Mladen Tomich, Daniel H. Fine, and David H. Figurski "The TadV Protein of Actinobacillus actinomycetemcomitans Is a Novel Aspartic Acid Prepilin Peptidase Required for Maturation of the Flp1 Pilin and TadE and TadF Pseudopilins" J. Bacteriol. 188 (19): 6899. 2006 | |
The tad locus of Actinobacillus actinomycetemcomitans encodes genes for the biogenesis of Flp pili, which allow the bacterium to adhere tenaciously to surfaces and form strong biofilms. Although tad (tight adherence) loci are widespread among bacterial and archaeal species, very little is known about the functions of the individual components of the Tad secretion apparatus. Here we characterize the mechanism by which the pre-Flp1 prepilin is processed to the mature pilus subunit. We demonstrate that the tadV gene encodes a prepilin peptidase that is both necessary and sufficient for proteolytic maturation of Flp1. TadV was also found to be
required for maturation of the TadE and TadF pilin-like proteins, which we term pseudopilins. Using sitedirected mutagenesis, we show that processing of pre-Flp1, pre-TadE, and pre-TadF is required for biofilm formation. Mutation of a highly conserved glutamic acid residue at position +5 of Flp1, relative to the cleavage site, resulted in a processed pilin that was blocked in assembly. In contrast, identical mutations in TadE or TadF had no effect on biofilm formation, indicating that the mechanisms by which Flp1 pilin and the pseudopilins function are distinct. We also determined that two conserved aspartic acid residues in TadV are critical for function of the prepilin peptidase. Together, our results indicate that the A. actinomycetemcomitans TadV protein is a member of a novel subclass of nonmethylating aspartic acid prepilin peptidases.