Preprints (publication date not yet finalized)

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

  • November 2008

Acehan D, Petzold C, Gumper I, Sabatini DD, Muller EJ, Cowin P, Stokes DL. Plakoglobin Is Required for Effective Intermediate Filament Anchorage to Desmosomes. J Invest Dermatol 2008 Nov; 128 (11):2665-75. Epub 2008 May 22. nyu.gif
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.

  • October 2008

Cheung J, Hendrickson WA. Crystal structures of C4-dicarboxylate ligand complexes with sensor domains of histidine kinases DcuS and DctB. J Biol Chem. 2008 Oct 31; 283 (44):30256-65. Epub 2008 Aug 12. ColumbiaUniversityintheCityofNewYorkcopy.gif
Two-component signaling systems allow bacteria to adapt to changing environments. Typically, a chemical or other stimulus is detected by the periplasmic sensor domain of a transmembrane histidine kinase sensor, which in turn relays a signal through a phosphotransfer cascade to the cognate cytoplasmic response regulator. Such systems lead ultimately to changes in gene expression or cell motility. Mechanisms of ligand binding and signal transduction through the cell membrane in histidine kinases are not fully understood. In an effort to further understand such processes, we have solved the crystal structures of the periplasmic sensor domains of Escherichia coli DcuS and of Vibrio cholerae DctB in complex with the respective cognate ligands, malate and succinate. Both proteins are involved in the regulation of the transport and metabolism of C4-dicarboxylates, but they are not highly related by sequence similarity. Our work reveals that despite disparate sizes, both structures contain a similar characteristic α/β PDC (PhoQ- DcuS- CitA) sensor-domain fold and display similar modes of ligand binding, suggesting similar mechanisms of function.

Eryilmaz E, Benach J, Su M, Seetharaman J, Dutta K, Wei H, Gottlieb P, Hunt JF, Ghose R.J Mol Biol. Structure and Dynamics of the P7 Protein from the Bacteriophage φ12. J Mol Biol 2008 Oct 3; 382 (2):402-22. Epub 2008 Jul 11. cuny.gif ColumbiaUniversityintheCityofNewYorkcopy.gif
Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multiprotein polymerase complex (PX) to replicate and transcribe the viral genome. Though the structures of the polymerase and ATPase components of the cystoviral PX are known and their functional behavior understood to a large extent, no atomic-resolution structural information is available for the major capsid protein P1 that defines the overall structure and symmetry of the viral capsid and the essential protein P7. Towards obtaining a complete structural and functional understanding of the cystoviral PX, we have obtained the structure of P7 from the cystovirus φ 12 at a resolution of 1.8 . The N-terminal core region (1-129) of P7 forms a novel homodimeric &alpha' / β -fold with structural similarities with BRCT domains implicated in multiple protein-protein interactions in DNA repair proteins. Our results combined with the known role of P7 in stabilizing the nucleation complex during capsid assembly hints towards its participation in key protein-protein interactions within the cystoviral PX. Additionally, we have found through solution NMR studies that the C-terminal tail of P7 (130-169) that is essential for virus viability, though highly disordered, contains a nascent helix. We demonstrate for the very first time, through NMR titrations, that P7 is capable of interacting with RNA. We find that both the Nterminal core and the dynamic C-terminus tail of P7 play a role in RNA recognition leading to a significant reduction of the degree of disorder in the C-terminal tail. Given the requirement of P7 in maintaining genome packaging efficiency and transcriptional fidelity, our data suggest a central biological role for P7/RNA interactions. Keywords: Bacteriophage, cystovirus, RNA polymerase complex, structural protein, proteinnucleic acid interactions.

Xie J, Reverdatto S, Frolov A, Hoffmann R, Burz DS, Shekhtman A. Structural basis for pattern recognition by the receptor for advanced glycation end products (RAGE). J Biol Chem. 2008 Oct 3; 283 (40):27255-69. Epub 2008 Jul 30. PMID: 18667420 suny.gif
The receptor for advanced glycated end products (RAGE) is a multiligand receptor that is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders and inflammatory responses. The ability of RAGE to recognize advanced glycated end products (AGEs) formed by nonenzymatic glycoxidation of cellular proteins places RAGE in the category of pattern recognition receptors. The structural mechanism of AGE recognition was an enigma due to the diversity of chemical structures found in AGE modified proteins. Here, using NMR spectroscopy we showed that the immunoglobulin V-type domain of RAGE is responsible for recognizing various classes of AGEs. Three distinct surfaces of the V domain were identified to mediate AGE-V domain interactions. They are located in the positively charged areas of the V domain. The first interaction surface consists of strand C and loop CC, the second interaction surface consists of strand C, strand F, and loop FG, and the third interaction surface consists of strand A and loop EF. The secondary structure elements of the interaction surfaces exhibit significant flexibility on the ms-s time scale. Despite highly specific AGE-V domain interactions, the binding affinity of AGEs for an isolated V domain is low, ~10 M. Using in-cell fluorescence resonance energy transfer (FRET) we show that RAGE is a constitutive oligomer on the plasma membrane. We propose that constitutive oligomerization of RAGE is responsible for recognizing patterns of AGE modified proteins with affinities less than 100 nM.

Edmund C. Schwartz, Alexander Shekhtman , Kaushik Dutta, Matthew R. Pratt, David Cowburn, Seth Darst and Tom W. Muir "Autoregulation of a Group 1 Bacterial Sigma Factor Involves the Formation of Region 1.1-Induced Compacted Structure " Chemistry and Biology 15 , 1091-1103 ru.gif
The bacterial σ factors fulfill a regulatory role in transcription by allowing the recognition of promoter DNA by RNA polymerase. The σ factors responsible for the majority of transcription initiation during log phase growth, known as the group 1 σ factors, are auto-regulated by an N-terminal sequence known as region 1.1. Here we report the solution structure of T. maritima σ-A region 1.1. We additionally demonstrate by using chemical crosslinking strategies that region 1.1 is in close proximity to the DNA binding domains of σ-A. We therefore propose that region 1.1 fulfils its regulatory role by holding the σ factor in compact structure that is unable to bind DNA.

Lee, YT; Dimitrova, YN; Schneider, G; Ridenour, WB; Bhattacharya, S; Soss, SE; Caprioli, RM; Filipek, A; Chazin, WJ "Structure of the S100A6 complex with a fragment from the C-terminal domain of Siah-1 interacting protein: A novel mode for S100 protein target recognition" ; BIOCHEMISTRY 47 (41):10921-10932 OCT 14 2008 Vanderbilt U;
Warsaw Inst.;
ABSTRACT: S100A6 is a member of the S100 subfamily of EF-hand Ca2+ binding proteins that has been shown to interact with calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP or SIP), a subunit of an SCFlike E3 ubiquitin ligase complex (SCF-TBL1) formed under genotoxic stress. SIP serves as a scaffold in this complex, linking the E2-recruiting module Siah-1 to the substrate-recruiting module Skp1-TBL1. A cell-based functional assay suggests that S100A6 modulates the activity of SCF-TBL1. The results from the cell-based experiments could be enhanced if it were possible to selectively inhibit S100A6-SIP interactions without perturbing any other functions of the two proteins. To this end, the structure of the S100A6-SIP complex was determined in solution by NMR and the strength of the interaction was characterized by isothermal titration calorimetry. In an initial step, the minimal S100A6 binding region in SIP was mapped to a 31-residue fragment (Ser189-Arg219) in the C-terminal domain. The structure of the S100A6-SIP(189-219) complex revealed that SIP(189-219) forms two helices, the first of which (Met193-Tyr200) interacts with S100A6 in a canonical binding mode. The second helix (Met207-Val216) lies over the S100A6 dimer interface, a mode of binding to S100A6 that has not previously been observed for any target bound to an S100 protein. A series of structurebased SIP mutations showed reduced S100A6 binding affinity, setting the stage for direct functional analysis of S100A6-SIP interactions.

  • September 2008

Shan B, Bhattacharya S, Eliezer D, Raleigh DP " The Low-pH Unfolded State of the C-Terminal Domain of the Ribosomal Protein L9 Contains Significant Secondary Structure in the Absence of Denaturant but Is No More Compact Than the Low-pH Urea Unfolded State." Biochemistry 2008 Sep 9; 47 (36):9565-73. Epub 2008 Aug 16 suny.gif
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PubMed
There is considerable interest in the properties of the unfolded states of proteins, particularly unfolded states which can be populated in the absence of high concentrations of denaturants. Interest in the unfolded state ensemble reflects the fact that it is the starting point for protein folding as well as the reference state for protein stability studies and can be the starting state for pathological aggregation. The unfolded state of the C-terminal domain (residues 58-149) of the ribosomal protein L9 (CTL9) can be populated in the absence of denaturant at low pH. CTL9 is a 92-residue globular alpha, beta protein. The low-pH unfolded state contains more secondary structure than the low-pH urea unfolded state, but it is not a molten globule. Backbone ( (1)H, (13)C, and (15)N) NMR assignments as well as side chain (13)C beta and (1)H beta assignments and (15)N R 2 values were obtained for the pH 2.0 unfolded form of CTL9 and for the urea unfolded state at pH 2.5. Analysis of the deviations of the chemical shifts from random coil values indicates that residues that comprise the two helices in the native state show a clear preference for adopting helical phi and psi angles in the pH 2.0 unfolded state. There is a less pronounced but nevertheless clear tendency for residues 107-124 to preferentially populate helical phi and psi values in the unfolded state. The urea unfolded state has no detectable tendency to populate any type of secondary structure even though it is as compact as the pH 2.0 unfolded state. Comparison of the two unfolded forms of CTL9 provides direct experimental evidence that states which differ significantly in their secondary structure can have identical hydrodynamic properties. This in turn demonstrates that global parameters such as R h or R g are very poor indicators of "random coil" behavior.

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 Sep; 1777 (9):1098-108. ColumbiaUniversityintheCityofNewYorkcopy.gif
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

Ling W, Eliav U, Navon G, Jerschow A. (2008) Chemical exchange saturation transfer by intermolecular double-quantum coherence. J Magn Reson, Sep; 194 (1):29-32. nyu.gif
A number of contrast enhancement effects based on the use of intermolecular multiple-quantum coherences, or distant dipolar field effects are known. This phenomenon is characterized by the dependence on the mth power of the initial magnetization (where m is the coherence order used). In this paper, we describe the contrast enhancement based on chemical exchange saturation transfer and NOE, which is achieved by the use of intermolecular double-quantum coherences (iDQC). The method was validated using clinically relevant systems based on glycosaminoglycans and a sample of cartilage tissue, showing that the CEST contrast, as well as, NOE are enhanced by iDQC.

Pellecchia M, Bertini I, Cowburn D, Dalvit C, Giralt E, Jahnke W, James TL, Homans SW, ... 'Perspectives on NMR in drug discovery" Nat Rev Drug Discov 2008 7 738-745 and 12 others
In the past decade, the potential of harnessing the ability of nuclear magnetic resonance (NMr) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. in this Perspective, we highlight some of the major applications of NMr in drug discovery, focusing on hit and lead generation, and provide a critical analysis of its current and potential utility.a technique comes of age.

  • August 2008

Rong P, Regatte RR, Jerschow A. (2008) Clean demarcation of cartilage tissue (23)Na by inversion recovery. J Magn Reson. Aug; 193 (2):207-9. nyu.gif
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.

Miloushev VZ, Bahna F, Ciatto C, Ahlsen G, Honig B, Shapiro L, Palmer AG 'Dynamic Properties of a Type II Cadherin Adhesive Domain: Implications for the Mechanism of Strand-Swapping of Classical Cadherins.' Structure 2008 Aug; 16 (8):1195-205 ColumbiaUniversityintheCityofNewYorkcopy.gif
Cadherin-mediated cell adhesion is achieved through dimerization of cadherin N-terminal extracellular (EC1) domains presented from apposed cells. The dimer state is formed by exchange of N-terminal β strands and insertion of conserved tryptophan indole side chains from one monomer into hydrophobic acceptor pockets of the partner molecule. The present work characterizes individual monomer and dimer states and the monomer-dimer equilibrium of the mouse Type II cadherin-8 EC1 domain using NMR spectroscopy. Limited picosecond- to-nanosecond timescale dynamics of the tryptophan indole moieties for both monomer and dimer states are consistent with well-ordered packing of the N-terminal b strands intramolecularly and intermolecularly, respectively. However, pronounced microsecond-to-millisecond timescale dynamics of the side chains are observed for the monomer but not the dimer state, suggesting that monomers transiently sample configurations in which the indole moieties are exposed. The results suggest possible kinetic mechanisms for EC1 dimerization.

Roxana E. Georgescu, . Olga Yurieva, Seung-Sup Kim, John Kuriyan, Xiang-Peng Kong, and Mike O'Donnell, (2008) Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp. Proc Natl Acad Sci U S A. 2008 Aug 12; 105 (32):11116-21. Epub 2008 Aug 4. ru.gif
DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli beta-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with beta and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to beta. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with beta, and that the beta-clamp may represent an antibiotic target.

  • July 2008

Wu J, Li W, Craddock BP, Foreman KW, Mulvihill MJ, Ji QS, Miller WT, Hubbard SR. (2008) Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor. EMBO J. Jul 23; 27 (14):1985-94. nyu.gif
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The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.

Lorieau JL, Day LA, McDermott AE. (2008) Conformational dynamics of an intact virus: Order parameters for the coat protein of Pf1 bacteriophage. Proc Natl Acad Sci U S A. 2008 Jul 29; 105 (30):10366-71. ColumbiaUniversityintheCityofNewYorkcopy.gif
This study has examined the atomic-level dynamics of the protein in the capsid of filamentous phage Pf1. This capsid consists of approximately 7,300 small subunits of only 46 aa in a helical array around a highly extended, circular single-stranded DNA molecule of 7,349 nt. Measurements were made of site-specific, solid-state NMR order parameters, S, the values which are dimensionless quantities between 0 (mobile) and 1 (static) that characterize the amplitudes of molecular bond angular motions that are faster than microseconds. It was found that the protein subunit backbone is very static, and of particular interest, it appears to be static at residues glycine 15 and glutamine 16 where it had been previously thought to be mobile. In contrast to the backbone, several side chains display large-amplitude angular motions. Side chains on the virion exterior that interact with solvent are highly mobile, but surprisingly, the side chains of residues arginine 44 and lysine 45 near the DNA deep in the interior of the virion are also highly mobile. The large-amplitude dynamic motion of these positively charged side chains in their interactions with the DNA were not previously expected. The results reveal a highly dynamic aspect of a DNA-protein interface within a virus.

Burz DS, Shekhtman A. (2008) In-cell biochemistry using NMR spectroscopy. PLoS ONE. 2008 Jul 2; 3 (7):e2571. PMID: 18626516 suny.gif
Biochemistry and structural biology are undergoing a dramatic revolution. Until now, mostly in vitro techniques have been used to study subtle and complex biological processes under conditions usually remote from those existing in the cell. We developed a novel in-cell methodology to post-translationally modify interactor proteins and identify the amino acids that comprise the interaction surface of a target protein when bound to the post-translationally modified interactors. Modifying the interactor proteins causes structural changes that manifest themselves on the interacting surface of the target protein and these changes are monitored using in-cell NMR. We show how Ubiquitin interacts with phosphorylated and nonphosphorylated components of the receptor tyrosine kinase (RTK) endocytic sorting machinery: STAM2 (Signal-transducing adaptor molecule), Hrs (Hepatocyte growth factor regulated substrate) and the STAM2-Hrs heterodimer. Ubiquitin binding mediates the processivity of a large network of interactions required for proper functioning of the RTK sorting machinery. The results are consistent with a weakening of the network of interactions when the interactor proteins are phosphorylated. The methodology can be applied to any stable target molecule and may be extended to include other post-translational modifications such as ubiquitination or sumoylation, thus providing a long-awaited leap to high resolution in cell biochemistry.

Dutta, K; Cox, CJ; Basavappa, R; Pascal, SM (2008) N-15 relaxation studies of apo-Mts1: A dynamic S100 protein BIOCHEMISTRY 47 (29):7637-7647 JUL 22 2008
Mts1 is a member of the S100 family of EF-hand calcium-binding proteins. Like most S100 proteins, Mts1 exists as a dimer in solution and contains one canonical and one pseudo-EF-hand motif per monomer, each of which consists of two R helices connected by a loop capable of coordinating a calcium ion. The backbone dynamics of murine apo-Mts1 homodimer have been examined by nuclear magnetic resonance spectroscopy. Longitudinal and transverse relaxation data and steady-state 1H-15N nuclear Overhauser effects were analyzed using model-free formalism. The extracted global correlation time is 9.94 ns. Results indicate that the protein backbone is most rigid at the dimer interface, made up of helices 1 and 4 from each monomer with mean S2 (Savg 2 ) values ∼0.9, flanked by helices 2 and 3 with lower Savg 2 values of 0.84 and 0.77, respectively. Each calcium-binding site along with the hinge joining the two EF-hands and the N- and C-termini are considerably more flexible than the dimer interface on a range of time scales and more flexible than the corresponding regions of other S100 proteins studied to date. As the hinge and the C-terminal tail are believed to interact with target proteins, these dynamic characteristics may have implications for Mts1 activity.

Qi Zhang, Hanudatta S. Atreya, Douglas E. Kamen, Mark E. Girvin, Thomas Szyperski (2008) GFT projection NMR based resonance assignment of membrane proteins: application to subunit c of E. coli F1F0 ATP synthase in LPPG micelles J Biomol NMR 2008 40 :157163 suny.gif
G-matrix FT projection NMR spectroscopy was employed for resonance assignment of the 79-residue subunit c of the Escherichia coli F1F0 ATP synthase embedded in micelles formed by lyso palmitoyl phosphatidyl glycerol (LPPG). Five GFT NMR experiments, that is, (3,2)D HNNCO, L-(4,3)D HNNCabCa?, L-(4,3)D HNN(CO)CabCa, (4,2)D HACA(CO)NHN and (4,3)D HCCH, were acquired along with simultaneous 3D 15N, 13Caliphatic, 13Caromatic-resolved [1H,1H]-NOESY with a total measurement time of*43 h. Data analysis resulted in sequence specific assignments for all routinely measured backbone and 13Cb shifts, and for 97% of the side chain shifts. Moreover, the use of two G2FT NMR experiments, that is, (5,3)D HN{N,CO}{CabCa} and (5,3)D {CabCa} {CON}HN, was explored to break the very high chemical shift degeneracy typically encountered for membrane proteins. It is shown that the 4D and 5D spectral information obtained rapidly from GFT and G2FT NMR experiments enables one to efficiently obtain (nearly) complete resonance assignments of membrane proteins.

George W. Wagner and Boris Itin Comment on "27AI. 47,49Ti. 31P. and 13C MlAS? NMR Study of VX: GD. and DD Reactions with Nanoosize Al03, Conventional Al03 and Ti02 and Aluminum and Titanium Metal" J. Phys. Chem. C 218， 112， 9962 ECBC

  • June 2008

Liu Q, Dansereau JT, Puttamadappa SS, Shekhtman A, Derbyshire V, Belfort M. (2008) Role of the interdomain linker in distance determination for remote cleavage by homing endonuclease I-TevI. J Mol Biol. 2008 Jun 20; 379 (5):1094-106 suny.gif wads.gif
I-TevI is a modular intron-encoded endonuclease, consisting of an Nterminal catalytic domain and a C-terminal DNA-binding domain, joined by a 75 amino acid linker. This linker can be divided into three regions, starting at the N terminus: the deletion-intolerant (DI) region; the deletiontolerant (DT) region; and a zinc finger, which acts as a distance determinant for cleavage. To further explore linker function, we generated deletion and substitution mutants that were tested for their preference to cleave at a particular distance or at the correct sequence. Our results demonstrate that the I-TevI linker is multi-functional, a property that sets it apart from junction sequences in most other proteins. First, the linker DI region has a role in I-TevI cleavage activity. Second, the DT linker region participates in distance determination, as evident from DT mutants that display a phenotype similar to that of the zinc-finger mutants in their selection of a cleavage site. Finally, NMR analysis of a freestanding 56 residue linker segment showed an unstructured stretch corresponding to the DI region and a portion of the DT region, followed by a β-strand corresponding to the remainder of the DT region and containing a key distance-determining arginine, R129. Mutation of this arginine to alanine abolished distance determination and disrupted the β-strand, indicating that the structure of the DT linker region has a role in cleavage at a fixed distance.

  • Mar 2008

Wilson RC and Pata JD (2008) Structural Insights into the Generation of Single-Base Deletions by the Y Family DNA Polymerase Dbh. Molecular Cell 29 :767-779 wads.gif
Dbh is a Y family translesion DNA polymerase that accurately bypasses some damaged forms of deoxyguanosine, but also generates single-base deletion errors at frequencies of up to 50%, in specific hot spot sequences. We describe preinsertion binary, insertion ternary, and postinsertion binary crystal structures of Dbh synthesizing DNA after making a single-base deletion. The skipped template base adopts an extrahelical conformation stabilized by interactions with the C-terminal domain of the enzyme. DNA translocation and positioning of the next templating base at the active site, with space opposite to accommodate incoming nucleotide, occur independently of nucleotide binding, incorporation, and pyrophosphate release. We also show that Dbh creates single-base deletions more rapidly when the skipped base is located two or three bases upstream of the nascent base pair than when it is directly adjacent to the templating base, indicating that Dbh predominantly creates single-base deletions by template slippage rather than by dNTPstabilized misalignment.

  • April 2007

George w. 刑Tagner，时Lawrence R. Procell,t and Shekar Munavalli:j:, (2007) "27AI, 47,49Ti, 31p, and 13C MAS NMR Study of VX, GD, and HD Reactions with Nanosize Al03, Conventional Al03 and Ti02, and Aluminum and Titanium Metal " J. Phys. Chem. C 2007, 111 , 17564一17569 ECBC
Reactions of VX, GD, and HD with Al03, Ti02 (anatase and rutile) , aluminum, and titanium metal powders have been studied by 27AI, 47,49Ti, 31p, and 13C MAS NMR. VX, GD, and HD hydrolyze on both nanosize and conventional Al03. A significant droplet size effect on the reaction kinetics is observed. For VX and GD, 27Al and 31p MAS NMR detect the formation of aluminum phosphonate complexes. Similarly, GD hydrolysis on Ti02 yields titanium phosphonate species as detected by 31p MAS NMR. Attempts at obtaining 47.49Ti NMR spectra of these species and those of titanium phosphonate model compounds at 14 T were marginally successful. 47,49Ti NMR spectra were obtainable for anatase and titanium metal; thus, severe second order quadrupolar line broadening is suspected for the titanium phosphonate complexes. 47,49Ti NMR spectra obtained for anatase at high magnetic field (17.5 and 21 T) showed anticipated improvement in peak width and resolution. GD reacted with aluminum and titanium powder in the presence of water results in acid dissolution of the metals and the formation of their respective metal phosphonates.

Yangsheng Zhou ; Debashish Ray; Yiwei Zhao; Hongping Dong; Suping Ren; Zhong Li; Yi Guo; Kristen A. Bernard; Pei-Yong Sh; and Hongmin Li (2007) Structure and Function of Flavivirus NS5 Methyltransferase J Virol. 81 (8), 38913903 wads.gif
The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA-->m7GpppA-->m7GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 A resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

  • February 2007

LM Wang, YW Zhao, Zh. Li, Y. Guo, D.M. Kranz, W Mourad, H. Li (2007), Structure of a complete ternary complex between TCR, superantigen, and Class II MHC molecule, Nature Structure & Molecular Biology 14, 169-171. wads.gif
Superantigens (SAgs) trigger the massive activation of T cells by simultaneous interactions with MHC and TCR receptors, leading to human diseases. Here we present the first crystal structure, at 2.5-A resolution, of a complete ternary complex between a SAg and its two receptors, HLA-DR1/HA and TCR. The most striking finding is that the SAg Mycoplasma arthritidis mitogen, unlike others, has direct contacts not only with TCR Vb but with TCR Va.

  • January 2007

Devrim Acehan;, Yang Xu;, David L. Stokes; and Michael Schlame; Comparison of lymphoblast mitochondria from normal subjects and patients with Barth syndrome using electron microscopic tomography Lab Invest. 2007 January; 87 (1):4048. nyu.gif PMC
Barth syndrome (BTHS) is a mitochondrial disorder that is caused by mutations in the tafazzin gene, which affects phospholipid composition. To determine whether this defect leads to alterations in the internal three-dimensional organization of mitochondrial membranes, we applied electron microscopic tomography to lymphoblast mitochondria from BTHS patients and controls. Tomograms were formed from 50 nm and 150 nm sections of chemically fixed lymphoblasts and the data were used to manually segment volumes of relevant structural details. Normal lymphoblast mitochondria contained well aligned, lamellar cristae with slot-like junctions to the inner boundary membrane. In BTHS, mitochondrial size was more variable and the total mitochondrial volume per cell increased, mainly due to clusters of fragmented mitochondria inside nuclear invaginations. However, mitochondria showed reduced cristae density, less cristae alignment, and inhomogeneous cristae distribution. Three-dimensional reconstruction of BTHS mitochondria revealed zones of adhesion of the opposing inner membranes, causing obliteration of the intracrista space. We found small isolated patches of adhesion as well as extended adhesion zones, resulting in sheets of collapsed cristae packaged in multiple concentric layers. We also found large tubular structures (diameter 30-150 nm) that appeared to be derivatives of the adhesion zones. The data suggest that mitochondrial abnormalities of BTHS involve adhesions of inner mitochondrial membranes with subsequent collapse of the intracristae space.

  • February 2006

Yi Guo,a Zhong Li, Sandra J. Van Vranken, and Hongmin Li (2006) A single point mutation changes the crystallization behavior of Mycoplasma arthritidis-derived mitogen. Acta Crystallograph F62, 238-41. Epub Feb 10. wads.gif
Mycoplasma arthritidis-derived mitogen (MAM) functions as a conventional superantigen (SAg). Although recombinant MAM has been crystallized by the hanging-drop vapour-diffusion method, the crystals diffracted poorly to only 5.0 resolution, with large unit-cell parameters a = 163.8, b = 93.0, c = 210.9 , β = 93.7 in the monoclinic space group P21. Unit-cell content analysis revealed that as many as 24 molecules could be present in the asymmetric unit. Systematic alanine mutagenesis was applied in order to search for mutants that give crystals of better quality. Two mutants, L50A and K201A, were crystallized under the same conditions as wild-type MAM (MAMwt). Crystals of the L50A mutant are isomorphous with those of MAMwt, while a new crystal form was obtained for the K201 mutant, belonging to the cubic space group P4132 with unit-cell parameters a = b = c = 181.9 . Diffraction data were collected to 3.6 and 2.8 resolution from crystals of the MAM L50A and K201A mutants, respectively. Molecular-replacement calculations suggest the presence of two molecules in the asymmetric unit for the MAM K201A mutant crystal, resulting in a V M of 5.0 Da−1 and a solvent content of 75%. An interpretable electron-density map for the MAM K201A mutant crystal was produced using the molecular-replacement method.

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

Highlights of NYSBC associated publications through 2004

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History: r14 - 06 Sep 2008 - 10:00:37 - DavidCowburn
 
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