Preprints (publication date not yet finalized)

Rong P, Regatte RR, Jerschow A. (2008) Clean demarcation of cartilage tissue (23)Na by inversion recovery. J Magn Reson. Epub May 1 nyu.gif Library Link
Monitoring the sodium concentration in vivo using (23)Na MRI can be an important tool for assessing the onset of tissue disorders. Practical clinical (23)Na MRI methods furthermore often do not allow one to use sufficiently small voxel sizes such that only the tissue of interest is seen, but a large signal contamination can arise from sodium in synovial fluid. Here we demonstrate that applying an inversion recovery (IR) technique allows one to distinctly select either the cartilage-bound or the free sodium for visualization in an image. The results are validated both ex vivo and in vivo.

Acehan D, Petzold C, Gumper I, Sabatini DD, Muller EJ, Cowin P, Stokes DL. (2008) Plakoglobin Is Required for Effective Intermediate Filament Anchorage to Desmosomes. J Invest Dermatol Epub May 22. nyu.gif
Library Link
Desmosomes are adhesive junctions that provide mechanical coupling between cells. Plakoglobin (PG) is a major component of the intracellular plaque that serves to connect transmembrane elements to the cytoskeleton. We have used electron tomography and immunolabeling to investigate the consequences of PG knockout on the molecular architecture of the intracellular plaque in cultured keratinocytes. Although knockout keratinocytes form substantial numbers of desmosome-like junctions and have a relatively normal intercellular distribution of desmosomal cadherins, their cytoplasmic plaques are sparse and anchoring of intermediate filaments is defective. In the knockout, beta-catenin appears to substitute for PG in the clustering of cadherins, but is unable to recruit normal levels of plakophilin-1 and desmoplakin to the plaque. By comparing tomograms of wild type and knockout desmosomes, we have assigned particular densities to desmoplakin and described their interaction with intermediate filaments. Desmoplakin molecules are more extended in wild type than knockout desmosomes, as if intermediate filament connections produced tension within the plaque. On the basis of our observations, we propose a particular assembly sequence, beginning with cadherin clustering within the plasma membrane, followed by recruitment of plakophilin and desmoplakin to the plaque, and ending with anchoring of intermediate filaments, which represents the key to adhesive strength.

Huang L, McDermott AE. (2008) Partial site-specific assignment of a uniformly (13)C, (15)N enriched membrane protein, light-harvesting complex 1 (LH1), by solid state NMR. Biochim Biophys Acta Epub Jan 26 ColumbiaUniversityintheCityofNewYorkcopy.gif link
Partial site-specific assignments are reported for the solid state NMR spectra of light-harvesting complex 1, a 160 kDa integral membrane protein. The assignments were derived from 600 MHz (15)N-(13)CO-(13)Calpha and (15)N-(13)Calpha-(13)CX correlation spectra, using uniformly (13)C, (15)N enriched hydrated material, in an intact and precipitated form. Sequential assignments were verified using characteristic (15)N-(13)Calpha-(13)Cbeta side chain chemical shifts observed in 3D experiments. Tertiary contacts found in 2D DARR spectra of the selectively (13)C enriched sample provided further confirmatory evidence for the assignments. The assignments include the region of the Histidine ligands binding the Bacteriochlorophyll chromophore. The chemical shifts of Calpha and Cbeta resonances indicated the presence of typical alpha-helical secondary structure, consistent with previous studies

Articles published in the last few months, or not previously reported

  • June 2008

Zeng L, Yap KL, Ivanov AV, Wang X, Mujtaba S, Plotnikova O, Rauscher Iii FJ, Zhou MM. (2008) Structural insights into human KAP1 PHD finger-bromodomain and its role in gene silencing. Nat Struct Mol Biol., 6, 626-33. mssm.gif Library Link
The tandem PHD finger-bromodomain, found in many chromatin-associated proteins, has an important role in gene silencing by the human co-repressor KRAB-associated protein 1 (KAP1). Here we report the three-dimensional solution structure of the tandem PHD finger-bromodomain of KAP1. The structure reveals a distinct scaffold unifying the two protein modules, in which the first helix, alpha(Z), of an atypical bromodomain forms the central hydrophobic core that anchors the other three helices of the bromodomain on one side and the zinc binding PHD finger on the other. A comprehensive mutation-based structure-function analysis correlating transcriptional repression, ubiquitin-conjugating enzyme 9 (UBC9) binding and SUMOylation shows that the PHD finger and the bromodomain of KAP1 cooperate as one functional unit to facilitate lysine SUMOylation, which is required for KAP1 co-repressor activity in gene silencing. These results demonstrate a previously unknown unified function for the tandem PHD finger-bromodomain as an intramolecular small ubiquitin-like modifier (SUMO) E3 ligase for transcriptional silencing.

Bhattacharya S, Zhang H, Debnath AK, Cowburn D. (2008) Solution structure of a hydrocarbon stapled peptide inhibitor in complex with monomeric C-terminal domain of HIV-1 capsid. J Biol Chem, 283, 16274-8.
NY Blood Center
The HIV-1 capsid protein plays a critical role in virus core particle assembly and is an important target for novel therapeutic strategies. In a previous study we characterized the binding affinity of a hydrocarbon stapled helical peptide, NYAD-1 for the capsid protein (Kd ~ 1 µM) and demonstrated its ability to penetrate the cell membrane. In cell based assays, NYAD-1 colocalized with the Gag polyprotein at the plasma membrane and disrupted the formation of mature and immature virus particles in vitro systems. Here, we complement the cellular and biochemical data with structural characterization of the interactions between the capsid and a soluble peptide analogue, NYAD-13. Solution NMR methods were used to determine a high resolution structure of the complex between the inhibitor and a monomeric form of the C-terminal domain of the capsid protein (mCA-CTD). The intermolecular interactions are mediated by the packing of hydrophobic side-chains at the buried interface and unperturbed by the presence of the olefinic chain on the solvent exposed surface of the peptide. Supported by GM66354, GM47021

Ferrage F, Piserchio A, Cowburn D, Ghose R. (2008) On the Measurement of 15N-{1H} Nuclear Overhauser Effects. J Mag Res, 192, 302-313. cuny.gif link
Accurate quantification of the 15N-{1H} steady-state NOE is central to current methods for the elucidation of protein backbone dynamics on the fast, sub-nanosecond timescale. This experiment is highly susceptible to systematic errors arising from multiple sources. The nature of these errors and their effects on the determined NOE ratio is evaluated by a detailed analysis of the spin dynamics during the pair of experiments used to measure this ratio and possible improvements suggested. The experiment that includes 1H irradiation, is analyzed in the framework of Average Liouvillian Theory and a modified saturation scheme that generates a stable steady state and eliminates the need to completely saturate 1H nuclei, is presented. The largest source of error, however, in 1H-dilute systems at ultra-high fields is found to be an overestimation of the steady-state NOE value as a consequence of the incomplete equilibration of the magnetization in the so-called reference experiment. The use of very long relaxation delays is usually an effective, but time consuming, solution. Here, we introduce an alternative reference experiment, designed for larger, deuterated systems, that uses the fastest relaxing component of the longitudinal magnetization a closer approximation to the equilibrium state for shorter relaxation delays. The utility of the modified approach is illustrated through simulations on realistic spin-systems over a wide range of timescales and experimentally verified using a perdeuterated sample of human ubiquitin. GM66354 , GM47021 supported.

Paleologou KE, Schmid AW, Rospigliosi CC, Kim HY, Lamberto GR, Fredenburg RA, Lansbury PT Jr, Fernandez CO, Eliezer D, Zweckstetter M, Lashuel HA. (2008) Phosphorylation at S129, but not the phosphomimics S129E/D inhibits the fibrilization of α -synuclein. J Biol Chem, 283, 16895-16905. EPL Laussane weil.gif MPI Goettingen; CNICT, Rosaria, Argentina; Harvard Library link
α-Synuclein (α-syn) phosphorylation at serine 129 is characteristic of Parkinson's disease (PD) and related α-synulceinopathies. However, whether phosphorylation promotes or inhibits α-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of α-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of S129 phosphorylation, we compared the biochemical, structural and membrane binding of wild type α-syn to those of the phosphorylation mimics (S129E, S129D) as well as the in vitro phosphorylated α-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at S129 increases the conformational flexibility of α-syn and inhibits its fibrillogenesis in vitro, but does not perturb its membrane-conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern α-syn aggregation and toxicity in PD and related α-synulceinopathies.

  • May 2008

Shen Y, Tong L. (2008) Structural Evidence for Direct Interactions between the BRCT Domains of Human BRCA1 and a Phospho-peptide from Human ACC1. Biochemistry, 47, 5767-73. ColumbiaUniversityintheCityofNewYorkcopy.gif PDB in process
The tandem BRCA1 C-terminal (BRCT) domains are phospho-serine/threonine recognition modules essential for the function of BRCA1. Recent studies suggest that acetyl-CoA carboxylase 1 (ACC1), an enzyme with crucial roles in de novo fatty acid biosynthesis and lipogenesis and essential for cancer cell survival, may be a novel binding partner for BRCA1, through interactions with its BRCT domains. We report here the crystal structure at 3.2 A resolution of human BRCA1 BRCT domains in complex with a phospho-peptide from human ACC1 (p-ACC1 peptide, with the sequence 1258-DSPPQ-pS-PTFPEAGH-1271), which provides molecular evidence for direct interactions between BRCA1 and ACC1. The p-ACC1 peptide is bound in an extended conformation, located in a groove between the tandem BRCT domains. There are recognizable and significant structural differences to the binding modes of two other phospho-peptides to these domains, from BACH1 and CtIP, even though they share a conserved pSer-Pro-(Thr/Val)-Phe motif. Our studies establish a framework for understanding the regulation of lipid biosynthesis by BRCA1 through its inhibition of ACC1 activity, which could be a novel tumor suppressor function of BRCA1.

Cheung J, Bingman CA, Reyngold M, Hendrickson WA, Waldburger CD. (2008) Crystal structure of a functional dimer of the PhoQ sensor domain. J Biol Chem, 283, 13762-70. ColumbiaUniversityintheCityofNewYorkcopy.gif
The PhoP-PhoQ two-component system is a well studied bacterial signaling system that regulates virulence and stress response. Catalytic activity of the histidine kinase sensor protein PhoQ is activated by low extracellular concentrations of divalent cations such as Mg2+, and subsequently the response regulator PhoP is activated in turn through a classic phosphotransfer pathway that is typical in such systems. The PhoQ sensor domains of enteric bacteria contain an acidic cluster of residues (EDDDDAE) that has been implicated in direct binding to divalent cations. We have determined crystal structures of the wild-type Escherichia coli PhoQ periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues (QNNNNAQ). The PhoQ domain structure is similar to that of DcuS and CitA sensor domains, and this PhoQ-DcuS-CitA (PDC) sensor fold is seen to be distinct from the superficially similar PAS domain fold. Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg50' and Asp179 and with nickel ions bound to aspartate residues in the acidic cluster. The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis. The mutant structure has an alternative, non-physiological dimeric association.

Xiong X, Cui P, Hossain S, Xu R, Warner B, Guo X, An X, Debnath A, Cowburn D, Kotula L. (2008) . Allosteric Inhibition of the nonMyristoylated c-Abl Tyrosine Kinase by Phosphopeptides Derived from Abi1/Hssh3bp1. BBA Molecular Cell Research, 1783, 737-47. NY Blood Center
Here we report c-Abl kinase inhibition mediated by a phosphotyrosine located in trans in the c-Abl substrate, Abi1. The mechanism, pertinent to the nonmyristoylated c-Abl kinase, involves high affinity concurrent binding of the phosphotyrosine pY213 to the Abl SH2 domain and binding of a proximal PXXP motif to the Abl SH3 domain. Abi1 regulation of c-Abl in vivo appears to play a critical role, as demonstrated by inhibition of pY412 phosphorylation of the nonmyristoylated Abl by coexpression of Abi1. Pervanadate-induced c-Abl kinase activity was also reduced upon expression of the wild type Abi1 but not of the Y to F mutant in LNCaP cells that are naturally deficient in the regulatory pY213. Our findings suggest a novel mechanism by which Abl kinase is regulated in cells. GM47021 supported.

Malgieri G, Eliezer D. (2008) Structural effects of Parkinson's disease linked DJ-1 mutations. Protein Sci., 17, 855-68. weil.gif library link
Mutations in the protein DJ-1 are associated with familial forms of Parkinson's disease, indicating that DJ-1 may be involved in pathways related to the etiology of this disorder. Here we have used solution state NMR and circular dichroism spectroscopies to evaluate the extent of structural perturbations associated with five different Parkinson's disease linked DJ-1mutations: L166P, E64D, M26I, A104T, and D149A. Comparison of the data with those obtained for the wild-type protein shows that the L166P mutation leads to severe and global destabilization and unfolding of the protein structure, while the structure of the E64D mutation, as expected, is nearly unperturbed. Interestingly, the remaining three mutants all show different degrees of structural perturbation, which are accompanied by a reduction in the thermodynamic stability of the protein. The observed structural and thermodynamic differences are likely to underlie any functional variations between these mutants and the wild type, which in turn are likely responsible for the pathogenicity of these mutations.

  • April 2008

Liu D, Xu R, Dutta K, Cowburn D. (2008) N-terminal cysteinyl proteins can be prepared using thrombin cleavage. FEBS letts , 582, 1163-7.
Expressed protein ligation – which allows native proteins to be selectively linked together by a normal peptide bond in an aqueous environment – has emerged as a powerful technique. The technique requires the formation of a C-terminal α-thioester and an N-terminal Cys. An N-terminal Cys can be formed by enzymatic cleavage, commonly using the Factor Xa and TEV proteases. We show that thrombin can be used for the formation of N-terminal Cys, providing another choice of reagents for expressed protein ligation. Proteins with N-terminal Cys can be obtained by the convenient modification of vectors changing the putative thrombin cleavage site LVPRG to LVPRC. Two example protein domains (Csk and Abl tyrosine kinase domain) with N-terminal Cys are demonstrated using this method. The use of thrombin protease to generate N-terminal Cys overcomes some of the limitations of existing methods, making it generally useful for expressed protein ligation and other biotechnological applications. GM47021 supported.

Yang J, Tasayco ML, Polenova T. (2008) Magic Angle Spinning NMR Experiments for Structural Studies of Differentially Enriched Protein Interfaces and Protein Assemblies. J Am Chem Soc, 30, 5798-807. U. Delaware cuny.gif Library link
Protein-protein interactions play vital roles in numerous biological processes. These interactions often result in formation of insoluble and noncrystalline protein assemblies. Solid-state NMR spectroscopy is rapidly emerging as a premier method for structural analysis of such systems. We introduce a family of two-dimensional magic angle spinning (MAS) NMR experiments for structural studies of differentially isotopically enriched protein assemblies. Using 1-73( (13)C, (15)N)/74-108( (15)N) labeled thioredoxin reassembly, we demonstrate that dipolar dephasing followed by proton-assisted heteronuclear magnetization transfer yields long-range (15)N- (13)C correlations arising exclusively from the interfaces formed by the pair of differentially enriched complementary fragments of thioredoxin. Incorporation of dipolar dephasing into the (15)N proton-driven spin diffusion and into the (1)H- (15)N FSLG-HETCOR sequences permits (1)H and (15)N resonance assignments of the 74-108( (15)N) enriched C-terminal fragment of thioredoxin alone. The differential isotopic labeling scheme and the NMR experiments demonstrated here allow for structural analysis of both the interface and each interacting protein. Isotope editing of the magnetization transfers results in spectral simplification, and therefore larger protein assemblies are expected to be amenable to these experiments.

George EA, Novick RP, Muir TW. (2008) Cyclic Peptide Inhibitors of Staphylococcal Virulence Prepared by Fmoc-Based Thiolactone Peptide Synthesis. J Am Chem Soc, 130, 4914-24. ru.gif nyu.gif link
Virulence factor production in Staphylococcus aureus is largely under the control of the accessory gene regulator (agr) quorum sensing system. There are four agr groups, all of which exhibit bacterial interference: each agr type synthesizes a cyclic autoinducing peptide (AIP) with a distinct sequence that activates its cognate AgrC receptor and inhibits activation of others. To better understand inhibitory AIP-AgrC interactions, we aimed to identify the minimal molecular determinants required to inhibit both non-cognate and cognate receptors. This minimization of the AIP pharmacophore also may have therapeutic relevance as the use of native AIPs to block virulence of non-cognate agr strains can prevent the establishment of an infection in vivo. We synthesized and evaluated the inhibitory activities of 10 AIP derivatives based on a truncated AIP analogue that inhibits all four agr types. To carry out the rapid, parallel synthesis of these peptides, we employed a new linker for Fmoc-based thioester peptide synthesis. Our results identify key structural elements that are necessary for AgrC inhibition and reveal key differences between non-cognate and cognate inhibitory requirements.

Gupta YK, Nair DT, Wharton RP, Aggarwal AK. (2008) Structures of Human Pumilio with Noncognate RNAs Reveal Molecular Mechanisms for Binding Promiscuity. Structure, 16, 549-557. mssm.gif
HHMI, Duke
Pumilio is a founder member of the evolutionarily conserved Puf family of RNA-binding proteins that control a number of physiological processes in eukaryotes. A structure of human Pumilio (hPum) Puf domain bound to a Drosophila regulatory sequence showed that each Puf repeat recognizes a single nucleotide. Puf domains in general bind promiscuously to a large set of degenerate sequences, but the structural basis for this promiscuity has been unclear. Here, we describe the structures of hPum Puf domain complexed to two noncognate RNAs, CycB(reverse) and Puf5. In each complex, one of the nucleotides is ejected from the binding surface, in effect, acting as a "spacer." The complexes also reveal the plasticity of several Puf repeats, which recognize noncanonical nucleotides. Together, these complexes provide a molecular basis for recognition of degenerate binding sites, which significantly increases the number of mRNAs targeted for regulation by Puf proteins in vivo.

Zhong J, Frases S, Wang H, Casadevall A, Stark RE. (2008) Following Fungal Melanin Biosynthesis with Solid-State NMR: Biopolymer Molecular Structures and Possible Connections to Cell-Wall Polysaccharides. Biochemistry, 47, 4701-4710 cuny.gif aeco.gif Library link
Melanins serve a variety of protective functions in plants and animals, but in fungi such as Cryptococcus neoformans they are also associated with virulence. A recently developed solid-state nuclear magnetic resonance (NMR) strategy, based on the incorporation of site-specific (13)C-enriched precursors into melanin, followed by spectroscopy of both powdered and solvent-swelled melanin ghosts, was used to provide new molecular-level insights into fungal melanin biosynthesis. The side chain of an l-dopa precursor was shown to cyclize and form a proposed indole structure in C. neoformans melanin, and modification of the aromatic rings revealed possible patterns of polymer chain elongation and cross-linking within the biopolymer. Mannose supplied in the growth medium was retained as a beta-pyranose moiety in the melanin ghosts even after exhaustive degradative and dialysis treatments, suggesting the possibility of tight binding or covalent incorporation of the pigment into the polysaccharide fungal cell walls. In contrast, glucose was scrambled metabolically and incorporated into both polysaccharide cell walls and aliphatic chains present in the melanin ghosts, consistent with metabolic use as a cellular nutrient as well as covalent attachment to the pigment. The prominent aliphatic groups reported previously in several fungal melanins were identified as triglyceride structures that may have one or more sites of chain unsaturation. These results establish that fungal melanin contains chemical components derived from sources other than l-dopa polymerization and suggest that covalent linkages between l-dopa-derived products and polysaccharide components may serve to attach this pigment to cell wall structures.

Forouhar F, Abashidze M, Xu H, Grochowski L L, Seetharaman J, Hussain M, Kuzin A, Chen Y, Zhou W, Xiao R, Acton T B, Montelione G T, Galinier A, White R H,Tong L. (2008) Molecular insights into the biosynthesis of the F420 coenzyme. J Biol Chem, 283, 1183211840. NESG; ColumbiaUniversityintheCityofNewYorkcopy.gif Rutgers; CNRS, Marseille
Coenzyme F420, a hydride carrier, is found in archaea and some bacteria and has crucial roles in methanogenesis, antibiotics biosynthesis, DNA repair, and activation of antitubercular compounds. CofD, 2-phospho-L-lactate transferase, catalyzes the last step in the biosynthesis of F420-0 (F420 without polyglutamate), by transferring the lactyl phosphate (LP) moiety of lactyl (2) diphospho-(5')guanosine (LPPG) to 7,8-didemethyl-8-hydroxy-5-deazariboflavin ribitol (Fo). CofD is highly conserved among F420-producing organisms, and weak sequence homologs are also found in non-F420-producing organisms. This superfamily does not share any recognizable sequence conservation with other proteins. Here we report the first crystal structures of CofD-the free enzyme and two ternary complexes, with Fo and Pi or with Fo and GDP-from Methanosarcina mazei. The active site is located at the C-terminal end of a Rossmann-fold core, and three large insertions make significant contributions to the active site and dimer formation. The observed binding modes of Fo and GDP can explain known biochemical properties of CofD, and are also supported by our binding assays. The structures provide significant molecular insights into the biosynthesis of the F420 coenzyme. Large structural differences in the active site region of the non-F420-producing CofD homologs suggest that they catalyze a different biochemical reaction.

Zeng L, Zhang Q, Gerona-Navarro G, Moshkina N, Zhou MM. (2008) Structural Basis of Site-Specific Histone Recognition by the Bromodomains of Human Coactivators PCAF and CBP/p300. Structure, 16 , 643-52. mssm.gif
Histone lysine acetylation is central to epigenetic control of gene transcription. Bromodomains of chromosomal proteins function as acetyl-lysine (Kac) binding domains. However, how bromodomains recognize site-specific histones remains unanswered. Here, we report three three-dimensional solution structures of the bromodomains of the human transcriptional coactivators CREB-binding protein (CBP) and p300/CBP-associated factor (PCAF) bound to peptides derived from histone acetylation sites at lysines 36 and 9 in H3, and lysine 20 in H4. From structural and biochemical binding analyses, we determine consensus histone recognition by the bromodomains of PCAF and CBP, which represent two different subgroups of the bromodomain family. Through bromodomain residues in the ZA and BC loops, PCAF prefers acetylation sites with a hydrophobic residue at (Kac+2) position and a positively charged or aromatic residue at (Kac+3), whereas CBP favors bulky hydrophobic residues at (Kac+1) and (Kac+2), a positively charged residue at (Kac-1), and an aromatic residue at (Kac-2).

Owen GR, Acehan D, Derr KD, Rice WJ, Stokes DL. (2008) Cryoelectron tomography of isolated desmosomes. Biochem Soc Trans, 36, 173-9. nyu.gif
Desmosomes are a complex assembly of protein molecules that form at the cell surface and mediate cell-cell adhesion. Much is known about the composition of desmosomes and there is an established consensus for the location of and interactions between constituent proteins within the assembly. Furthermore, X-ray crystallography has determined atomic structures of isolated domains from several constituent proteins. Nevertheless, there is a lack of understanding about the architecture of the intact assembly and the physical principles behind the adhesive strength of desmosomes therefore remain vague. We have used electron tomography to address this problem. In previous work, we investigated the in situ structure of desmosomes from newborn mouse skin preserved by freeze-substitution and imaged in resin-embedded thin sections. In our present work, we have isolated desmosomes from cow snout and imaged them in the frozen unstained state. Although not definitive, the resulting images provide support for the irregular groupings of cadherin molecules seen previously in mouse skin.

Liu J, Wang D, Zheng Q, Lu M, Arora PS. (2008) Atomic structure of a short alpha-helix stabilized by a main chain hydrogen-bond surrogate. J Am Chem Soc., 130, 4334-7 weil.gif nyu.gif
Herein we report the 1.15 A X-ray crystal structure of a short alpha-helix in which the N-terminal i and i + 4 main chain hydrogen bond is replaced with a carbon-carbon bond. The structure shows that the hydrogen-bond surrogate (HBS) derived alpha-helix truly resembles the structure of canonical alpha-helices and provides unequivocal support for our helix nucleation strategy.

Banerjee S, Huber T, and Sakmar TP. (2008) Rapid Incorporation of Functional Rhodopsin into Nanoscale Apolipoprotein Bound Bilayer (NABB) Particles. Journal of Molecular Biology, 377, 1067-1081. ru.gif  
Human apolipoprotein A-I (apo A-I) and its engineered constructs form discoidal lipid bilayers upon interaction with lipids in vitro. We now report the cloning, expression, and purification of apo A-I derived from zebrafish (Danio rerio), which combines with phospholipids to form similar discoidal bilayers and may prove to be superior to human apo A-I constructs for rapid reconstitution of seven-transmembrane helix receptors into nanoscale apolipoprotein bound bilayers (NABBs). We characterized NABBs by gel-filtration chromatography, native polyacrylamide gradient gel electrophoresis, UV-visible photobleaching difference spectroscopy, and fluorescence spectroscopy. We used electron microscopy to determine the stoichiometry and orientation of rhodopsin (rho)-containing NABBs prepared under various conditions and correlated stability and signaling efficiency of rho in NABBs with either one or two receptors. We discovered that the specific activity of G protein coupling for single rhos sequestered in individual NABBs was nearly identical with that of two rhos per NABB under conditions where stoichiometry and orientation could be inferred by electron microscopy imaging. Thermal stability of rho in NABBs was superior to that of rho in various commonly used detergents. We conclude that the NABB system using engineered zebrafish apo A-I is a native-like membrane mimetic system for G-protein-coupled receptors and discuss strategies for rapid incorporation of expressed membrane proteins into NABBs.

Zhang H, Zhao Q, Bhattacharya S, Waheed A, Tong X, Hong A, Heck S, Curreli F, Goger M, Cowburn D, Freed E, Debnath A. (2008) A Cell-penetrating Helical Peptide as a Potential HIV-1 Inhibitor. J Miol Biol, 378, 565-80 NY Blood Center
The HIV-1 capsid protein plays a critical role in virus core particle assembly and is an important target for novel therapeutic strategies. In a previous study we characterized the binding affinity of a hydrocarbon stapled helical peptide, NYAD-1 for the capsid protein (Kd ~ 1 µM) and demonstrated its ability to penetrate the cell membrane. In cell based assays, NYAD-1 colocalized with the Gag polyprotein at the plasma membrane and disrupted the formation of mature and immature virus particles in vitro systems. Here, we complement the cellular and biochemical data with structural characterization of the interactions between the capsid and a soluble peptide analogue, NYAD-13. Solution NMR methods were used to determine a high resolution structure of the complex between the inhibitor and a monomeric form of the C-terminal domain of the capsid protein (mCA-CTD). The intermolecular interactions are mediated by the packing of hydrophobic side-chains at the buried interface and unperturbed by the presence of the olefinic chain on the solvent exposed surface of the peptide. The results of the structural analysis provide valuable insight into the determinants for high affinity and selective inhibitors for HIV-1 particle assembly. GM66354 supported.

  • March 2008

Koehnke J, Jin X, Trbovic N, Katsamba PS, Brasch J, Ahlsen G, Scheiffele P, Honig B, Palmer AG 3rd, Shapiro L. (2008) Crystal Structures of beta-Neurexin 1 and beta-Neurexin 2 Ectodomains and Dynamics of Splice Insertion Sequence 4. Structure, 16, 410-21. ColumbiaUniversityintheCityofNewYorkcopy.gif  
Presynaptic neurexins (NRXs) bind to postsynaptic neuroligins (NLs) to form Ca(2+)-dependent complexes that bridge neural synapses. beta-NRXs bind NLs through their LNS domains, which contain a single site of alternative splicing (splice site 4) giving rise to two isoforms: +4 and Delta. We present crystal structures of the Delta isoforms of the LNS domains from beta-NRX1 and beta-NRX2, crystallized in the presence of Ca(2+) ions. The Ca(2+)-binding site is disordered in the beta-NRX2 structure, but the 1.7 A beta-NRX1 structure reveals a single Ca(2+) ion, approximately 12 A from the splice insertion site, with one coordinating ligand donated by a glutamic acid from an adjacent beta-NRX1 molecule. NMR studies of beta-NRX1+4 show that the insertion sequence is unstructured, and remains at least partially disordered in complex with NL. These results raise the possibility that beta-NRX insertion sequence 4 may function in roles independent of neuroligin binding.

Wu J, Tseng YD, Xu CF, Neubert TA, White MF, Hubbard SR. (2008) Structural and biochemical characterization of the KRLB region in insulin receptor substrate-2. Nat Struct Mol Biol, 15, 251-8. nyu.gif
Insulin receptor substrates 1 and 2 (IRS1 and -2) are crucial adaptor proteins in mediating the metabolic and mitogenic effects of insulin and insulin-like growth factor 1. These proteins consist of a pleckstrin homology domain, a phosphotyrosine binding domain and a C-terminal region containing numerous sites of tyrosine, serine and threonine phosphorylation. Previous yeast two-hybrid studies identified a region unique to IRS2, termed the kinase regulatory-loop binding (KRLB) region, which interacts with the tyrosine kinase domain of the insulin receptor. Here we present the crystal structure of the insulin receptor kinase in complex with a 15-residue peptide from the KRLB region. In the structure, this segment of IRS2 is bound in the kinase active site with Tyr628 positioned for phosphorylation. Although Tyr628 was phosphorylated by the insulin receptor, its catalytic turnover was poor, resulting in kinase inhibition. Our studies indicate that the KRLB region functions to limit tyrosine phosphorylation of IRS2.

  • January 2008

Nguyen TT, Brown S, Fedorov AA, Fedorov EV, Babbitt PC, Almo SC, Raushel FM. (2008). At the periphery of the amidohydrolase superfamily: Bh0493 from Bacillus halodurans catalyzes the isomerization of D-galacturonate to D-tagaturonate. Biochemistry 47, 1194-206. aeco.gif  
The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. One of the most divergent members of this superfamily, uronate isomerase from Escherichia coli, catalyzes the isomerization of d-glucuronate to d-fructuronate and d-galacturonate to d-tagaturonate and is the only uronate isomerase in this organism. A gene encoding a putative uronate isomerase in Bacillus halodurans (Bh0705) was identified based on sequence similarity to uronate isomerases from other organisms. Kinetic evidence indicates that Bh0705 is relatively specific for the isomerization of d-glucuronate to d-fructuronate, confirming this functional assignment. Despite a low sequence identity to all other characterized uronate isomerases, phylogenetic and network-based analysis suggests that a second gene in this organism, Bh0493, is also a uronate isomerase, although it is an outlier in the group, with <20% sequence identity to any other characterized uronate isomerase from another species. The elucidation of the X-ray structure at a resolution of 2.0 A confirms that Bh0493 is a member of the amidohydrolase superfamily with conserved residues common to other members of the uronate isomerase family. Functional characterization of this protein shows that unlike Bh0705, Bh0493 can utilize both d-glucuronate and d-galacturonate as substrates. In B. halodurans, Bh0705 is found in an operon for the metabolism of d-glucuronate, whereas Bh0493 is in an operon for the metabolism of d-galacturonate. These results provide the first identification of a uronate isomerase that operates in a pathway distinct from that for d-glucuronate. While most organisms that contain this pathway have only one gene for a uronate isomerase, sequence analysis and operon context show that five other organisms also appear to have two genes and one organism appears to have three genes for this activity.

  • November 2007

Cunha L, Kuti M, Bishop DF, Mezei M, Zeng L, Zhou MM, Desnick RJ. (2007) Human uroporphyrinogen III synthase: NMR-based mapping of the active site.' Proteins, 71, 855-873. mssm.gif
Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex.

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Highlights of NYSBC associated publications through 2004

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History: r11 - 17 Mar 2008 - 16:15:32 - DavidCowburn
 
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