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Showing papers in "Nature Structural & Molecular Biology in 1999"


Journal ArticleDOI
TL;DR: Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours and is demonstrated with examples of a few recently solved structures.
Abstract: In protein crystallography, much time and effort are often required to trace an initial model from an interpretable electron density map and to refine it until it best agrees with the crystallographic data. Here, we present a method to build and refine a protein model automatically and without user intervention, starting from diffraction data extending to resolution higher than 2.3 A and reasonable estimates of crystallographic phases. The method is based on an iterative procedure that describes the electron density map as a set of unconnected atoms and then searches for protein-like patterns. Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours. We demonstrate the power of the method with examples of a few recently solved structures.

2,463 citations


Journal ArticleDOI
TL;DR: It is discovered that in λ-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN, which makes use of the melting transition to prepare single poly(dG-dC) andpoly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence.
Abstract: Atomic force microscope-based single-molecule force spectroscopy was employed to measure sequence-dependent mechanical properties of DNA by stretching individual DNA double strands attached between a gold surface and an AFM tip. We discovered that in lambda-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN. During this transition the DNA is split into single strands that fully recombine upon relaxation. The sequence dependence was investigated in comparative studies with poly(dG-dC) and poly(dA-dT) DNA. Both the B-S and the melting transition occur at significantly lower forces in poly(dA-dT) compared to poly(dG-dC). We made use of the melting transition to prepare single poly(dG-dC) and poly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence. The unzipping of these hairpins directly revealed the base pair-unbinding forces for G-C to be 20 +/- 3 pN and for A-T to be 9 +/- 3 pN.

792 citations


Journal ArticleDOI
TL;DR: The HCV NS5B apoenzyme structure reported here can accommodate a template:primer duplex without global conformational changes, supporting the hypothesis that this structure is essentially preserved during the reaction pathway.
Abstract: Various classes of nucleotidyl polymerases with different transcriptional roles contain a conserved core structure. Less is known, however, about the distinguishing features of these enzymes, particularly those of the RNA-dependent RNA polymerase class. The 1. 9 A resolution crystal structure of hepatitis C virus (HCV) nonstructural protein 5B (NS5B) presented here provides the first complete and detailed view of an RNA-dependent RNA polymerase. While canonical polymerase features exist in the structure, NS5B adopts a unique shape due to extensive interactions between the fingers and thumb polymerase subdomains that serve to encircle the enzyme active site. Several insertions in the fingers subdomain account for intersubdomain linkages that include two extended loops and a pair of antiparallel alpha-helices. The HCV NS5B apoenzyme structure reported here can accommodate a template:primer duplex without global conformational changes, supporting the hypothesis that this structure is essentially preserved during the reaction pathway. This NS5B template:primer model also allows identification of a new structural motif involved in stabilizing the nascent base pair.

782 citations


Journal ArticleDOI
TL;DR: The three-dimensional structure of CHS elucidates the chemical basis of plant polyketide biosynthesis and provides a framework for engineering CHS-like enzymes to produce new products.
Abstract: Chalcone synthase (CHS) is pivotal for the biosynthesis of flavonoid antimicrobial phytoalexins and anthocyanin pigments in plants. It produces chalcone by condensing one p-coumaroyl- and three malonyl-coenzyme A thioesters into a polyketide reaction intermediate that cyclizes. The crystal structures of CHS alone and complexed with substrate and product analogs reveal the active site architecture that defines the sequence and chemistry of multiple decarboxylation and condensation reactions and provides a molecular understanding of the cyclization reaction leading to chalcone synthesis. The structure of CHS complexed with resveratrol also suggests how stilbene synthase, a related enzyme, uses the same substrates and an alternate cyclization pathway to form resveratrol. By using the three-dimensional structure and the large database of CHS-like sequences, we can identify proteins likely to possess novel substrate and product specificity. The structure elucidates the chemical basis of plant polyketide biosynthesis and provides a framework for engineering CHS-like enzymes to produce new products.

619 citations


Journal ArticleDOI
TL;DR: The blockage of the pore suggests that the N–terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm, and this structure could provide a signaling pathway between the processes of ligand recognition and TonB–mediated transport.
Abstract: Integral outer membrane receptors for iron chelates and vitamin B12 carry out specific ligand transport against a concentration gradient. Energy for active transport is obtained from the proton-motive force of the inner membrane through physical interaction with TonB-ExbB-ExbD, an inner membrane complex. Here we report the crystal structure of an active transport, outer membrane receptor at 2.4 A resolution. Two distinct functional domains are revealed: (i) a 22-stranded beta-barrel that spans the outer membrane and contains large extracellular loops which appear to function in ligand binding; and (ii) a globular N-terminal domain that folds into the barrel pore, inhibiting access to the periplasm and contributing two additional loops for potential ligand binding. These loops could provide a signaling pathway between the processes of ligand recognition and TonB-mediated transport. The blockage of the pore suggests that the N-terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm.

570 citations


Journal ArticleDOI
TL;DR: The high-resolution, refined structures of eNOS and iNOS reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.
Abstract: Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 A resolution) and iNOS (2.25 A resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.

421 citations


Journal ArticleDOI
TL;DR: Folate derivatives protect wild-type and mutant E. coli enzymes against flavin loss, and protect human MTHFR and the A222V mutant against thermal inactivation, suggesting a mechanism by which folate treatment reduces homocysteine levels.
Abstract: Elevated plasma homocysteine levels are associated with increased risk for cardiovascular disease and neural tube defects in humans. Folate treatment decreases homocysteine levels and dramatically reduces the incidence of neural tube defects. The flavoprotein methylenetetrahydrofolate reductase (MTHFR) is a likely target for these actions of folate. The most common genetic cause of mildly elevated plasma homocysteine in humans is the MTHFR polymorphism A222V (base change C677-->T). The X-ray analysis of E. coli MTHFR, reported here, provides a model for the catalytic domain that is shared by all MTHFRs. This domain is a beta8alpha8 barrel that binds FAD in a novel fashion. Ala 177, corresponding to Ala 222 in human MTHFR, is near the bottom of the barrel and distant from the FAD. The mutation A177V does not affect Km or k(cat) but instead increases the propensity for bacterial MTHFR to lose its essential flavin cofactor. Folate derivatives protect wild-type and mutant E. coli enzymes against flavin loss, and protect human MTHFR and the A222V mutant against thermal inactivation, suggesting a mechanism by which folate treatment reduces homocysteine levels.

413 citations


Journal ArticleDOI
TL;DR: Kinetic analysis of mutations at 52 of the 57 residues in the src SH3 domain revealed that the transition state ensemble is even more polarized than suspected, and the similarity in folding mechanism of different SH3 domains and the similar hierarchy of structure formation observed in the experiments and the simulations can be largely accounted for by a simple native state topology-based model of protein folding energy landscapes.
Abstract: We use a combination of experiments, computer simulations and simple model calculations to characterize, first, the folding transition state ensemble of the src SH3 domain, and second, the features of the protein that determine its folding mechanism Kinetic analysis of mutations at 52 of the 57 residues in the src SH3 domain revealed that the transition state ensemble is even more polarized than suspected earlier: no single alanine substitution in the N-terminal 15 residues or the C-terminal 9 residues has more than a two-fold effect on the folding rate, while such substitutions at 15 sites in the central three-stranded β-sheet cause significant decreases in the folding rate Molecular dynamics (MD) unfolding simulations and ab initio folding simulations on the src SH3 domain exhibit a hierarchy of folding similar to that observed in the experiments The similarity in folding mechanism of different SH3 domains and the similar hierarchy of structure formation observed in the experiments and the simulations can be largely accounted for by a simple native state topology-based model of protein folding energy landscapes

392 citations


Journal ArticleDOI
TL;DR: The structure of mouse importin α has been determined and explains the regulatory switch between the cytoplasmic, high-affinity form, and the nuclear, low-Affinity form for NLS binding of the nuclear import receptor predicted by the current models of nuclear import.
Abstract: Importin α is the nuclear import receptor that recognizes classical monopartite and bipartite nuclear localization signals (NLSs). The structure of mouse importin α has been determined at 2.5 A resolution. The structure shows a large C-terminal domain containing armadillo repeats, and a less structured N-terminal importin β-binding domain containing an internal NLS bound to the NLS-binding site. The structure explains the regulatory switch between the cytoplasmic, high-affinity form, and the nuclear, low-affinity form for NLS binding of the nuclear import receptor predicted by the current models of nuclear import. Importin β conceivably converts the low- to high-affinity form by binding to a site overlapping the autoinhibitory sequence. The structure also has implications for understanding NLS recognition, and the structures of armadillo and HEAT repeats.

384 citations


Journal ArticleDOI
TL;DR: The 2 Å crystal structure reported here of the collagen-like model peptide, T3-785, provides the first visualization of how the sequence of collagen defines distinctive local conformational variations in triple-helical structure.
Abstract: The 2 A crystal structure reported here of the collagen-like model peptide, T3-785, provides the first visualization of how the sequence of collagen defines distinctive local conformational variations in triple-helical structure.

378 citations


Journal ArticleDOI
TL;DR: Two X-ray structures of CGTase complexes are determined, which give evidence for substrate distortion and the covalent character of the intermediate and for the first time show, in atomic detail, how catalysis in the α-amylase family proceeds by the concerted action of all active site residues.
Abstract: Cyclodextrin glycosyltransferase (CGTase) is an enzyme of the α-amylase family, which uses a double displacement mechanism to process α-linked glucose polymers. We have determined two X-ray structures of CGTase complexes, one with an intact substrate at 2.1 A resolution, and the other with a covalently bound reaction intermediate at 1.8 A resolution. These structures give evidence for substrate distortion and the covalent character of the intermediate and for the first time show, in atomic detail, how catalysis in the α-amylase family proceeds by the concerted action of all active site residues.

Journal ArticleDOI
TL;DR: The protein engineering analysis of the α-spectrin SH3 domain at three different stability conditions reveals a folding transition state structured around the distal loop β-hairpin and the 310-helix, suggesting a transition state ensemble with little conformational variability.
Abstract: The protein engineering analysis of the α-spectrin SH3 domain at three different stability conditions (pH 7.0, 3.5 and 2.5) reveals a folding transition state structured around the distal loop β-hairpin and the 310-helix. This region is impervious to overall changes in protein stability, suggesting a transition state ensemble with little conformational variability. Comparison with the Src SH3 domain (36% sequence homology) indicates that the transition state in this protein family may be conserved. Discrepancies at some positions can be rationalized in terms of the different interactions made by the different side chains in both domains. Bronsted plot analysis confirms the φ‡-U results and shows two folding subdomains for this small protein. These results, together with previous data on circular permutants of the α-spectrin SH3 domain, indicate that polypeptide topology and chain connectivity play a major role in the folding reaction of this protein family.

Journal ArticleDOI
TL;DR: The structures of functional peptides corresponding to the predicted channel-lining M2 segments of the nicotinic acetylcholine receptor and of a glutamate receptor of the NMDA subtype (NMDAR) were determined using solution NMR experiments on micelle samples, and solid-state N MR experiments on bilayer samples.
Abstract: The structures of functional peptides corresponding to the predicted channel-lining M2 segments of the nicotinic acetylcholine receptor (AChR) and of a glutamate receptor of the NMDA subtype (NMDAR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. Both M2 segments form straight transmembrane α-helices with no kinks. The AChR M2 peptide inserts in the lipid bilayer at an angle of 12° relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminal side of the membrane, which is assigned to be intracellular. A model built from these solid-state NMR data, and assuming a symmetric pentameric arrangement of M2 helices, results in a funnel-like architecture for the channel, with the wide opening on the N-terminal intracellular side.

Journal ArticleDOI
TL;DR: Cryo-electron microscopy has been used to visualize elongation factor G (EF-G) on the 70S ribosome in GDP and GTP states and large conformational changes have been identified in the ribosomes.
Abstract: Cryo-electron microscopy has been used to visualize elongation factor G (EF-G) on the 70S ribosome in GDP and GTP states. GTP hydrolysis is required for binding of all the domains of EF-G to the pretranslocational complex and for the completion of translocation. In addition, large conformational changes have been identified in the ribosome. The head of the 30S subunit shifts toward the L1 protein side, and the L7/L12 stalk becomes bifurcated upon EF-G binding. Upon GTP hydrolysis, the bifurcation is reversed and an arc-like connection is formed between the base of the stalk and EF-G.

Journal ArticleDOI
TL;DR: A case study presents the key concepts and experimental results that have led to the current understanding of cooperative oxygen binding by hemoglobin and concludes that the underlying simplicity of the two-state allosteric mechanism could not have been demonstrated without novel physical experiments and a rigorous quantitative analysis.
Abstract: The enormous success of structural biology challenges the physical scientist. Can biophysical studies provide a truly deeper understanding of how a protein works than can be obtained from static structures and qualitative analysis of biochemical data? We address this question in a case study by presenting the key concepts and experimental results that have led to our current understanding of cooperative oxygen binding by hemoglobin, the paradigm of structure function relations in multisubunit proteins. We conclude that the underlying simplicity of the two-state allosteric mechanism could not have been demonstrated without novel physical experiments and a rigorous quantitative analysis.

Journal ArticleDOI
TL;DR: NMR spectroscopy data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into close proximity and possibly leading to membrane fusion.
Abstract: SNARE (soluble NSF attachment protein receptor) proteins assemble into a stable complex essential for vesicle-membrane fusion. To further understand SNARE function we have used solution nuclear magnetic resonance (NMR) spectroscopy to characterize three assembly states of a yeast SNARE complex: first, the 'closed' conformation of Sso1; second, the binary complex of Sso1 and Sec9; and third, the ternary complex of Sso1, Sec9 and Snc1. Sec9 and Snc1 are unstructured in isolation. Sso1 likely consists of a four helix bundle formed by part of the C-terminal Hcore domain and the N-terminal H(A)H(B)H(C) domain, and this bundle is flanked on both sides by large flexible regions. Sso1 switches to an 'open' state when its Hcore domain binds Sec9. Conformational switching of the Hcore domain, via H(A)H(B)H(C), may provide a key regulatory mechanism in SNARE assembly. Formation of binary and ternary complexes induces additional alpha-helical structure in previously unstructured regions. Our data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into close proximity and possibly leading to membrane fusion.

Journal ArticleDOI
TL;DR: Comparison of the rates of folding of AcP and four other proteins with the same topology, including ADA2h, supports the concept that the average distance in sequence between interacting residues (that is, the contact order) is an important determinant of the rate of protein folding.
Abstract: Muscle acylphosphatase (AcP) is a small protein that folds very slowly with two-state behavior. The conformational stability and the rates of folding and unfolding have been determined for a number of mutants of AcP in order to characterize the structure of the folding transition state. The results show that the transition state is an expanded version of the native protein, where most of the native interactions are partially established. The transition state of AcP turns out to be remarkably similar in structure to that of the activation domain of procarboxypeptidase A2 (ADA2h), a protein having the same overall topology but sharing only 13% sequence identity with AcP. This suggests that transition states are conserved between proteins with the same native fold. Comparison of the rates of folding of AcP and four other proteins with the same topology, including ADA2h, supports the concept that the average distance in sequence between interacting residues (that is, the contact order) is an important determinant of the rate of protein folding.

Journal ArticleDOI
TL;DR: The crystal structure of heme oxygenase-1 (HO-1) reported here reveals a novel helical fold with the heme sandwiched between two helices, allowing close contact between the helix backbone and heme in addition to providing flexibility for substrate binding and product release.
Abstract: Heme oxygenase catalyzes the first step in the oxidative degradation of heme. The crystal structure of heme oxygenase-1 (HO-1) reported here reveals a novel helical fold with the heme sandwiched between two helices. The proximal helix provides a heme iron ligand, His 25. Conserved glycines in the distal helix near the oxygen binding site allow close contact between the helix backbone and heme in addition to providing flexibility for substrate binding and product release. Regioselective oxygenation of the alpha-meso heme carbon is due primarily to steric influence of the distal helix.

Journal ArticleDOI
TL;DR: The first structure of a complex between p11 and its cognate peptide, the N-terminus of annexin II, as well as that of p11 alone are reported, suggesting a model for an oxidized form of other S100 proteins that have been found in the extracellular milieu.
Abstract: The aggregation and membrane fusion properties of annexin II are modulated by the association with a regulatory light chain called p11.p11 is a member of the S100 EF-hand protein family, which is unique in having lost its calcium-binding properties. We report the first structure of a complex between p11 and its cognate peptide, the N-terminus of annexin II, as well as that of p11 alone. The basic unit for p11 is a tight, non-covalent dimer. In the complex, each annexin II peptide forms hydrophobic interactions with both p11 monomers, thus providing a structural basis for high affinity interactions between an S100 protein and its target sequence. Finally, p11 forms a disulfide-linked tetramer in both types of crystals thus suggesting a model for an oxidized form of other S100 proteins that have been found in the extracellular milieu.

Journal ArticleDOI
TL;DR: This finding suggests that the regulatory effects of histone acetylation may be exerted by bromodomain-containing proteins.
Abstract: It has recently been demonstrated that bromodomains — motifs found in several eukaryotic transcription factors — bind to acetyl-lysine, a modification of histones that is important for transcription. This finding suggests that the regulatory effects of histone acetylation may be exerted by bromodomain-containing proteins.

Journal ArticleDOI
TL;DR: The crystal structure at 2.2 Å resolution of a complex between TRAIL and the extracellular region of DR5 is reported, showing that TRAIL forms a central homotrimer around which three DR5 molecules bind.
Abstract: TRAIL, an apoptosis inducing ligand, has at least four cell surface receptors including the death receptor DR5. Here we report the crystal structure at 2.2 A resolution of a complex between TRAIL and the extracellular region of DR5. TRAIL forms a central homotrimer around which three DR5 molecules bind. Radical differences in the surface charge of the ligand, together with variation in the alignment of the two receptor domains confer specificity between members of these ligand and receptor families. The existence of a switch mechanism allowing variation in receptor domain alignment may mean that it is possible to engineer receptors with multiple specificities by exploiting contact positions unique to individual receptor-ligand pairs.

Journal ArticleDOI
TL;DR: The 2.0 Å X–ray crystal structure of a SAM domain homodimer from the intracellular region of the EphA4 receptor tyrosine kinase reveals a mode of dimerization that is predicted to be shared amongst the SAM domains ofThe Eph receptor tyosine kinases and possibly other SAM domain containing proteins.
Abstract: The sterile alpha motif (SAM) domain is a novel protein module of ~70 amino acids that is found in a variety of signaling molecules including tyrosine and serine/threonine protein kinases, cytoplasmic scaffolding and adaptor proteins, regulators of lipid metabolism, and GTPases as well as members of the ETS family of transcription factors. The SAM domain can potentially function as a protein interaction module through the ability to homo– and hetero–oligomerize with other SAM domains. This functional property elicits the oncogenic activation of chimeric proteins arising from translocation of the SAM domain of TEL to coding regions of the βPDGF receptor, Abl, JAK2 protein kinase and the AML1 transcription factor. Here we describe the 2.0 A X–ray crystal structure of a SAM domain homodimer from the intracellular region of the EphA4 receptor tyrosine kinase. The structure reveals a mode of dimerization that we predict is shared amongst the SAM domains of the Eph receptor tyrosine kinases and possibly other SAM domain containing proteins. These data indicate a mechanism through which an independently folding protein module can form homophilic complexes that regulate signaling events at the membrane and in the nucleus.

Journal ArticleDOI
TL;DR: The NMR-derived structure of the homodimeric core domain of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions, is presented, revealing the molecular assembly of two active sites within the dimeric kinase.
Abstract: Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223–289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.

Journal ArticleDOI
TL;DR: Differences in the relative size and shape of the MMP S1' pockets suggest that this pocket is a critical determinant of MMP inhibitor selectivity.
Abstract: The X-ray crystal structures of the catalytic domain of human collagenase-3 (MMP-13) and collagenase-1 (MMP-1) with bound inhibitors provides a basis for understanding the selectivity profile of a novel series of matrix metalloprotease (MMP) inhibitors. Differences in the relative size and shape of the MMP S1' pockets suggest that this pocket is a critical determinant of MMP inhibitor selectivity. The collagenase-3 S1' pocket is long and open, easily accommodating large P1' groups, such as diphenylether. In contrast, the collagenase-1 S1' pocket must undergo a conformational change to accommodate comparable P1' groups. The selectivity of the diphenylether series of inhibitors for collagenase-3 is largely determined by their affinity for the preformed S1' pocket of collagenase-3, as compared to the induced fit in collagenase-1.

Journal ArticleDOI
TL;DR: Structural similarities with cysteine-rich growth factors, taken together with its known growth-promoting properties, suggests the APP N-terminal domain could function as a growth factor in vivo.
Abstract: Amyloid precursor protein (APP) plays a central role in Alzheimer disease. A proteolytic-breakdown product of APP, called beta-amyloid, is a major component of the diffuse and fibrillar deposits found in Alzheimer diseased brains. The normal physiological role of APP remains largely unknown despite much work. A knowledge of its function will not only provide insights into the genesis of the disease but may also prove vital in the development of an effective therapy. Here we describe the 1.8 A resolution crystal structure of the N-terminal, heparin-binding domain of APP (residues 28-123), which is responsible, among other things, for stimulation of neurite outgrowth. The structure reveals a highly charged basic surface that may interact with glycosaminoglycans in the brain and an abutting hydrophobic surface that is proposed to play an important functional role such as dimerization or ligand binding. Structural similarities with cysteine-rich growth factors, taken together with its known growth-promoting properties, suggests the APP N-terminal domain could function as a growth factor in vivo.

Journal ArticleDOI
TL;DR: NMR relaxation experiments indicate that the backbone flexibility of mouse major urinary protein increases upon binding the hydrophobic mouse pheromone 2-sec-butyl-4,5-dihydrothiazole, which may represent a general mechanism to promote binding of very small ligands to macromolecules.
Abstract: For complexes between proteins and very small hydrophobic ligands, hydrophobic effects alone may be insufficient to outweigh the unfavorable entropic terms resulting from bimolecular association NMR relaxation experiments indicate that the backbone flexibility of mouse major urinary protein increases upon binding the hydrophobic mouse pheromone 2-sec-butyl-4,5-dihydrothiazole The associated increase in backbone conformational entropy of the protein appears to make a substantial contribution toward stabilization of the protein–pheromone complex This term is likely comparable in magnitude to other important free energy contributions to binding and may represent a general mechanism to promote binding of very small ligands to macromolecules

Journal ArticleDOI
TL;DR: The structure of calcium-bound calmodulin (Ca2+/CaM) complexed with a 26-residue peptide, corresponding to the CaM-binding domain of rat Ca2+ /CaM-dependent protein kinase kinase (CaMKK), has been determined by NMR spectroscopy.
Abstract: The structure of calcium-bound calmodulin (Ca2+/CaM) complexed with a 26-residue peptide, corresponding to the CaM-binding domain of rat Ca2+/CaM-dependent protein kinase kinase (CaMKK), has been determined by NMR spectroscopy In this complex, the CaMKK peptide forms a fold comprising an α-helix and a hairpin-like loop whose C-terminus folds back on itself The binding orientation of this CaMKK peptide by the two CaM domains is opposite to that observed in all other CaM–target complexes determined so far The N- and C-terminal hydrophobic pockets of Ca2+/CaM anchor Trp 444 and Phe 459 of the CaMKK peptide, respectively This 14-residue separation between two key hydrophobic groups is also unique among previously determined CaM complexes The present structure represents a new and distinct class of Ca2+/CaM target recognition that may be shared by other Ca2+/CaM-stimulated proteins

Journal ArticleDOI
TL;DR: The crystal structure of the kinetically trapped VIP2–NAD complex identifies the NAD binding cleft within the C-terminal enzymatic domain and provides a structural basis for understanding the targeting and catalysis of the medically and environmentally important binary toxins.
Abstract: A member of the Bacillus-produced vegetative insecticidal proteins (VIPs) possesses high specificity against the major insect pest, corn rootworms, and belongs to a class of binary toxins and regulators of biological pathways distinct from classical A-B toxins. The 1.5 A resolution crystal structure of the enzymatic ADP-ribosyltransferase component, VIP2, from Bacillus cereus reveals structurally homologous N- and C-terminal alpha/beta domains likely representing the entire class of binary toxins and implying evolutionary relationships between families of ADP-ribosylating toxins. The crystal structure of the kinetically trapped VIP2-NAD complex identifies the NAD binding cleft within the C-terminal enzymatic domain and provides a structural basis for understanding the targeting and catalysis of the medically and environmentally important binary toxins. These structures furthermore provide specific experimental results to help resolve paradoxes regarding the specific mechanism of ADP-ribosylation of actin by implicating ground state destabilization and nicotinamide product sequestration as the major driving forces for catalysis.

Journal ArticleDOI
Jun Wang1, Wei Wang1
TL;DR: Several possible simplified schemes from the relationship of minimized mismatch versus the number of residue types are found, including an optimal reduction with five types of residues that has the same form as the simplified palette of Baker and coworkers.
Abstract: What is the minimal number of residue types required to form a structured protein? This question is important for understanding protein modeling and design. Recently, an experimental finding by Baker and coworkers suggested a five-residue solution to this problem. We were motivated by their results and by the arguments of Wolynes to study reductions of protein representation based on the concept of mismatch between a reduced interaction matrix and the Miyazawa and Jernigan (MJ) matrix. We find several possible simplified schemes from the relationship of minimized mismatch versus the number of residue types (N = ~2–20). As a specific case, an optimal reduction with five types of residues has the same form as the simplified palette of Baker and coworkers. Statistical and kinetic features of a number of sequences are tested. Comparison of results from sequences with 20 residue types and their reduced representations indicates that the reduction by mismatch minimization is successful. For example, sequences with five types of residues have good folding ability and kinetic accessibility in model studies.

Journal ArticleDOI
TL;DR: The LukF structure illustrates how a channel–forming toxin masks protein–protein and protein–membrane interfaces prior to cell binding and assembly, and together with the α–hemolysin heptamer structure, they define the end points on the pathway of toxin assembly.
Abstract: Staphylococcal LukF, LukS, HγII, and α–hemolysin are self–assembling, channel–forming proteins related in sequence and function. In the α–hemolysin heptamer, the channel–forming β–strands and the amino latch make long excursions from the protomer core. Here we report the crystal structure of the water soluble form of LukF. In the LukF structure the channel–forming region folds into an amphipathic, three–strand β–sheet and the amino latch forms a β–strand extending a central β–sheet. The LukF structure illustrates how a channel–forming toxin masks protein–protein and protein–membrane interfaces prior to cell binding and assembly, and together with the α–hemolysin heptamer structure, they define the end points on the pathway of toxin assembly.