Preprints (publication date not yet finalized)

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 nyu.gif
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
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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.

Andrea Piserchio, Pravin A. Nair, Stewart Shuman and Ranajeet Ghose " Solution NMR Studies of Chlorella Virus DNA Ligase Adenylate" J. Mol. Biol. in press cuny.gif

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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 15N spin-relaxation and 15N H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (ps-ns) and slow (µs-ms) timescales, indicative of inter- and intra-domain 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.

Levin, E. J., M. Quick and M. Zhou (2009). "Crystal structure of a bacterial homologue of the kidney urea transporter. " Nature advance online publication. ColumbiaUniversityintheCityofNewYorkcopy.gif
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, ColumbiaUniversityintheCityofNewYorkcopy.gif
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

  • Dec 2009

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 weil.gif
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. aeco.gif
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.

  • Oct 2009

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. ColumbiaUniversityintheCityofNewYorkcopy.gif
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.

  • Sept. 2009

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 suny.gif ColumbiaUniversityintheCityofNewYorkcopy.gif
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. mskc.gif
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. ColumbiaUniversityintheCityofNewYorkcopy.gif
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. ColumbiaUniversityintheCityofNewYorkcopy.gif
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) cuny.gif
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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.

  • August 2009

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. ColumbiaUniversityintheCityofNewYorkcopy.gif

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 nyu.gif weil.gif
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.

  • June 2009

Pinar Akcora and Sanat K. Kumar "ANISOTROPIC SELF-ASSEMBLY OF NANOPARTICLES IN COMPOSITES" WO 2009078985 20090625 ColumbiaUniversityintheCityofNewYorkcopy.gif
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 ColumbiaUniversityintheCityofNewYorkcopy.gif
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.
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.

  • April 2009
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. cuny.gif
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. nyu.gif
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.

  • Oct 2006

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 ColumbiaUniversityintheCityofNewYorkcopy.gif
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.

List of publications by institution, possibly not yet including all above

Highlights of NYSBC associated publications through 2004

This topic: Main > ScientificReportDec09 > RecentPreprintsAndReprints > PapersAssociated
History: r16 - 31 Aug 2009 - 16:27:08 - DavidCowburn
 
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