Journal Articles

  • Mechanistic Basis of Plasmid-Specific DNA Binding of the F Plasmid Regulatory Protein, TraM
    [Nov 2014]

    Publication date: 11 November 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 22

    Author(s): Yun Peng , Jun Lu , Joyce J.W. Wong , Ross A. Edwards , Laura S. Frost , J.N. Mark Glover

    The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon–helix–helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.
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    Categories: Journal Articles
  • Structural Determinants in Prion Protein Folding and Stability
    [Nov 2014]

    Publication date: 11 November 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 22

    Author(s): Federico Benetti , Xevi Biarnés , Francesco Attanasio , Gabriele Giachin , Enrico Rizzarelli , Giuseppe Legname

    Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases, involving post-translational modifications of the cellular prion protein. Epidemiological studies on Creutzfeldt-Jakob disease, a prototype prion disorder, show a majority of cases being sporadic, while the remaining occurrences are either genetic or iatrogenic. The molecular mechanisms by which PrPC is converted into its pathological isoform have not yet been established. While point mutations and seeds trigger the protein to cross the energy barriers, thus causing genetic and infectious transmissible spongiform encephalopathies, respectively, the mechanism responsible for sporadic forms remains unclear. Since prion diseases are protein-misfolding disorders, we investigated prion protein folding and stability as functions of different milieus. Using spectroscopic techniques and atomistic simulations, we dissected the contribution of major structural determinants, also defining the energy landscape of prion protein. In particular, we elucidated (i) the essential role of the octapeptide region in prion protein folding and stability, (ii) the presence of a very enthalpically stable intermediate in prion-susceptible species, and (iii) the role of the disulfide bridge in prion protein folding.
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    Categories: Journal Articles
  • Insights into the Mechanisms of Membrane Curvature and Vesicle Scission by the Small GTPase Sar1 in the Early Secretory Pathway
    [Nov 2014]

    Publication date: 11 November 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 22

    Author(s): Hanaa Hariri , Nilakshee Bhattacharya , Kerri Johnson , Alex J. Noble , Scott M. Stagg

    The small GTPase protein Sar1 is known to be involved in both the initiation of COPII-coated vesicle formation and scission of the nascent vesicle from the endoplasmic reticulum. The molecular details for the mechanism of membrane remodeling by Sar1 remain unresolved. Here, we show that Sar1 transforms synthetic liposomes into structures of different morphologies including tubules and detached vesicles. We demonstrate that Sar1 alone is competent for vesicle scission in a manner that depends on the concentration of Sar1 molecules occupying the membrane. Sar1 molecules align on low-curvature membranes to form an extended lattice. The continuity of this lattice breaks down as the curvature locally increases. The smallest repeating unit constituting the ordered lattice is a Sar1 dimer. The three-dimensional structure of the Sar1 lattice was reconstructed by substituting spherical liposomes with galactoceramide lipid tubules of homogeneous diameter. These data suggest that Sar1 dimerization is responsible for the formation of constrictive membrane curvature. We propose a model whereby Sar1 dimers assemble into ordered arrays to promote membrane constriction and COPII-directed vesicle scission.
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    Categories: Journal Articles
  • How proteins knot their ties
    [Nov 2014]

    Publication date: Available online 1 November 2014
    Source:Journal of Molecular Biology

    Author(s): Franz X. Schmid







    Categories: Journal Articles
  • Mia40 combines thiol oxidase and disulfide isomerase activity to efficiently catalyze oxidative folding in mitochondria
    [Nov 2014]

    Publication date: Available online 1 November 2014
    Source:Journal of Molecular Biology

    Author(s): Johanna R. Koch , Franz X. Schmid

    Mia40 catalyzes disulfide bond formation in proteins in the mitochondrial intermembrane space. By using Cox17 as a natural substrate, we discovered that in the presence of Mia40 the formation of native disulfides is strongly favored. The catalytic mechanism of Mia40 involves a functional interplay between the chaperone site and the catalytic disulfide. Mia40 forms a specific native disulfide in Cox17 much more rapidly than other disulfides, in particular non native ones, which originates from the recently described high affinity for hydrophobic regions near target cysteines and the long life time of the mixed disulfide. In addition to its thiol oxidase function, Mia40 is active also as a disulfide reductase and isomerase. We found that species with inadvertently formed incorrect disulfides are rebound by Mia40 and reshuffled, revealing a proofreading mechanism that is steered by the conformational folding of the substrate protein.
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    Categories: Journal Articles
  • Short aggregation-prone peptide detectives: Finding proteins and truths about aggregation
    [Nov 2014]

    Publication date: Available online 1 November 2014
    Source:Journal of Molecular Biology

    Author(s): Tanja Mittag , Melissa R. Marzahn







    Categories: Journal Articles
  • The high-risk HPV16 E7 oncoprotein mediates interaction between the transcriptional coactivator CBP and the retinoblastoma protein pRb
    [Nov 2014]

    Publication date: Available online 1 November 2014
    Source:Journal of Molecular Biology

    Author(s): Ariane L. Jansma , Maria A. Martinez-Yamout , Rong Liao , Peiqing Sun , H. Jane Dyson , Peter E. Wright

    The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the CREB-binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high risk HPV16-E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.
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    Categories: Journal Articles
  • One more piece down to solve the III-A CRISPR puzzle
    [Nov 2014]

    Publication date: Available online 1 November 2014
    Source:Journal of Molecular Biology

    Author(s): Robert P. Hayes , Ailong Ke







    Categories: Journal Articles
  • Crystal structure of the Csm3-Csm4 subcomplex in the type III-A CRISPR-Cas interference complex
    [Nov 2014]

    Publication date: Available online 30 October 2014
    Source:Journal of Molecular Biology

    Author(s): Tomoyuki Numata , Hideko Inanaga , Chikara Sato , Takuo Osawa

    Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci play a pivotal role in the prokaryotic host defense system against invading genetic materials. The CRISPR loci are transcribed to produce CRISPR RNAs (crRNAs), which form interference complexes with CRISPR-associated (Cas) proteins to target the invading nucleic acid for degradation. The interference complex of the type III-A CRISPR-Cas system is composed of five Cas proteins (Csm1–Csm5) and a crRNA, and targets invading DNA. Here we show that the Csm1, Csm3, and Csm4 proteins from Methanocaldococcus jannaschii form a stable subcomplex. We also report the crystal structure of the M. jannaschii Csm3-Csm4 subcomplex at 3.1Å resolution. The complex structure revealed the presence of a basic concave surface around their interface, suggesting the RNA and/or DNA binding ability of the complex. A gel retardation analysis showed that the Csm3-Csm4 complex binds single stranded RNA in a non-sequence specific manner. Csm4 structurally resembles Cmr3, a component of the type III-B CRISPR-Cas interference complex. Based on bioinformatics, we constructed a model structure of the Csm1-Csm4-Csm3 ternary complex, which provides insights into its role in the Csm interference complex.
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    Categories: Journal Articles
  • Transport of L-glutamine, L-alanine, L-arginine and L-histidine by the neuron-specific Slc38a8 (SNAT8) in CNS
    [Nov 2014]

    Publication date: Available online 30 October 2014
    Source:Journal of Molecular Biology

    Author(s): Maria G.A. Hägglund , Sofie V. Hellsten , Sonchita Bagchi , Gaëtan Philippot , Erik Löfqvist , Victor C.O. Nilsson , Ingrid Almkvist , Edvin Karlsson , Smitha Sreedharan , Atieh Tafreshiha , Robert Fredriksson

    Glutamine transporters are important for regulating levels of glutamate and GABA in the brain. To date, six members of the SLC38 family (SNATs) have been characterized and functionally subdivided into System A (SNAT1, SNAT2 and SNAT4) and System N (SNAT3, SNAT5 and SNAT7). Here we present a first functional characterization of SLC38A8, one of the previous orphan transporters from the family and we suggest that the encoded protein should be named SNAT8 to adhere with the SNAT nomenclature. We show that SLC38A8 have preference for transporting L-glutamine, L-alanine, L-arginine, L-histidine, and L-aspartate using a Na+-dependent transport mechanism and that the functional characteristics of SNAT8 has highest similarity to the known System A transporters. We also provide a comprehensive CNS expression profile in mouse brain for the Slc38a8 gene and the SNAT8 protein. We show that Slc38a8 (SNAT8) is expressed in all neurons, both excitatory and inhibitory, in mouse brain using in situ hybridization and immunohistochemistry. Furthermore, proximity ligation assay show highly similar subcellular expression of SNAT7 and SNAT8. In conclusion, the neuronal SLC38A8 have a broad amino acid transport profile and is the first identified neuronal System A transporter. This suggests a key role of SNAT8 in the glutamine/glutamate(GABA) cycle in the brain.
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    Categories: Journal Articles
  • INO80-C and SWR-C: Guardians of the Genome
    [Nov 2014]

    Publication date: Available online 30 October 2014
    Source:Journal of Molecular Biology

    Author(s): Christian-Benedikt Gerhold , Michael H. Hauer , Susan M. Gasser

    The double membrane of the eukaryotic nucleus encapsulates the genome, constraining it to a nuclear sphere. Proteins, RNA-protein particles and artificial chromosome rings diffuse rapidly and freely throughout the nucleoplasm, while chromosomal loci move subdiffusively with varying degrees of constraint. In situ biochemical approaches and live imaging studies have revealed the existence of nuclear subcompartments that are enriched for specific chromatin states and/or enzymatic activities. This sequestration is thought to enhance the propagation or efficient establishment of heterochromatin, particularly when factors of limited abundance are involved. Implicit in the concept of compartmentation, is the notion that chromatin is able to move from one compartment to another. Indeed, in budding yeast, gene activation, repression, and the presence of persistent DNA double-strand breaks has each been shown to provoke subnuclear relocalization of chromatin. In some cases movement has been linked to the action of ATP-dependent chromatin remodeling complexes, more specifically to the Snf2-related ATPase containing complexes, SWR-C and INO80-C. Here we examine how these multi-subunit remodelers contribute to chromatin–based processes linked to the DNA damage response. Chromatin remodelers are able to alter the physical mobility of chromatin as they alter the compaction and organization of nucleosomes. Finally we review recent evidence that supports a role for yeast SWR-C and INO80-C in determining the subnuclear position of damaged domains, and recap the multiple ways in which these remodelers contribute to genomic integrity.
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    Categories: Journal Articles
  • Computational Design of Selective Peptides to Discriminate Between Similar PDZ Domains in an Oncogenic Pathway
    [Nov 2014]

    Publication date: Available online 30 October 2014
    Source:Journal of Molecular Biology

    Author(s): Fan Zheng , Heather Jewell , Jeremy Fitzpatrick , Jian Zhang , Dale F. Mierke , Gevorg Grigoryan

    Reagents that target protein-protein interactions to rewire signaling are of great relevance in biological research. Computational protein design may offer a means of creating such reagents on demand, but methods for encoding targeting selectivity are sorely needed. This is especially challenging when targeting interactions with ubiquitous recognition modules—e.g., PDZ domains, which bind C-terminal sequences of partner proteins. Here we consider the problem of designing selective PDZ inhibitor peptides in the context of an oncogenic signaling pathway, in which two PDZ domains (NHERF-2 PDZ2—N2P2 and MAGI-3 PDZ6—M3P6) compete for a receptor C-terminus to differentially modulate oncogenic activities. Because N2P2 increases tumorigenicity and M3P6 decreases it, we sought to design peptides that inhibit N2P2 without affecting M3P6. We developed a structure-based computational design framework that models peptide flexibility in binding, yet is efficient enough to rapidly analyze tradeoffs between affinity and selectivity. Designed peptides showed low-micromolar inhibition constants for N2P2 and no detectable M3P6 binding. Peptides designed for reverse discrimination bound M3P6 tighter than N2P2, further testing our technology. Experimental and computational analysis of selectivity determinants revealed significant indirect energetic coupling in the binding site. Successful discrimination between N2P2 and M3P6, despite their overlapping binding preferences, is highly encouraging for computational approaches to selective PDZ targeting, especially because design relied on a homology model of M3P6. Still, we demonstrate specific deficiencies of structural modeling that must be addressed to enable truly robust design. The presented framework is general and can be applied in many scenarios to engineer selective targeting.
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    Categories: Journal Articles
  • Interplay between E. coli DnaK, ClpB and GrpE during protein disaggregation
    [Nov 2014]

    Publication date: Available online 29 October 2014
    Source:Journal of Molecular Biology

    Author(s): Shannon M. Doyle , Shankar Shastry , Andrea N. Kravats , Yu-Hsuan Shih , Marika Miot , Joel R. Hoskins , George Stan , Sue Wickner

    The DnaK/Hsp70 chaperone system and ClpB/Hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. The teamwork is specific: E. coli DnaK interacts with E. coli ClpB and yeast Hsp70, Ssa1, interacts with yeast Hsp104. This interaction is between the M-domains of hexameric ClpB/Hsp104 and the DnaK/Hsp70 nucleotide-binding domain (NBD). To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomain IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Computational modeling of the DnaK-ClpB hexamer complex indicated that one DnaK monomer contacts two adjacent ClpB protomers simultaneously. The model and the experiments support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation.
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    Categories: Journal Articles
  • Quality Control in Eukaryotic Membrane Protein Overproduction
    [Nov 2014]

    Publication date: Available online 28 October 2014
    Source:Journal of Molecular Biology

    Author(s): Jennifer A. Thomas , Christopher G. Tate

    The overexpression of authentically folded eukaryotic membrane proteins in milligramme quantities is a fundamental prerequisite for structural studies. One of the most commonly used expression systems for the production of mammalian membrane proteins is the baculovirus expression system in insect cells. However, a detailed analysis by radioligand binding and comparative Western blotting of G protein-coupled receptors and a transporter produced in insect cells showed that a considerable proportion of the expressed protein was misfolded and incapable of ligand binding. In contrast, production of the same membrane proteins in stable inducible mammalian cell lines suggested that the majority was folded correctly. It was noted that detergent solubilisation of the misfolded membrane proteins using either digitonin or dodecylmaltoside was considerably less efficient than using sodium dodecyl sulfate or foscholine-12, whilst these detergents were equally efficient at solubilising correctly folded membrane proteins. This provides a simple and rapid test to suggest whether heterologously expressed mammalian membrane proteins are indeed correctly folded, without requiring radioligand binding assays. This will greatly facilitate the high-throughput production of fully functional membrane proteins for structural studies.
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    Categories: Journal Articles
  • Corrigendum to “Statistical Mechanics of Monod–Wyman–Changeux” [J Mol Biol 425 (9) (May 13 2013) 1433-1460]
    [Nov 2014]

    Publication date: Available online 25 October 2014
    Source:Journal of Molecular Biology

    Author(s): Sarah Marzen , Hernan G. Garcia , Rob Phillips







    Categories: Journal Articles
  • Editorial Board
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21









    Categories: Journal Articles
  • Contents List
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21









    Categories: Journal Articles
  • Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Andrew F. Taylor , Susan K. Amundsen , Miklos Guttman , Kelly K. Lee , Jie Luo , Jeffrey Ranish , Gerald R. Smith

    Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5′ GCTGGTGG 3′) during DNA unwinding, RecBCD's unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here, we identify, during RecBCD's DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small-angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with these two changes. We propose that, upon DNA binding, the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3′-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular “signal transduction” from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi's stimulation of recombination.
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    Categories: Journal Articles
  • Alteration of the C-Terminal Ligand Specificity of the Erbin PDZ Domain by Allosteric Mutational Effects
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Javier Murciano-Calles , Megan E. McLaughlin , Ariel Erijman , Yogesh Hooda , Nishant Chakravorty , Jose C. Martinez , Julia M. Shifman , Sachdev S. Sidhu

    Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.
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    Categories: Journal Articles
  • A Structural Portrait of the PDZ Domain Family
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Andreas Ernst , Brent A. Appleton , Ylva Ivarsson , Yingnan Zhang , David Gfeller , Christian Wiesmann , Sachdev S. Sidhu

    PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.
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    Categories: Journal Articles
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