Refined 1.8 A crystal structure of the lambda repressor-operator complex.

The crystal structure of the lambda repressor-operator complex has been refined to an R-factor of 18.9% at 1.8 A resolution. This refinement, using data collected at low temperature, has revealed the structure of the N-terminal arm and shows that the interactions of repressor with the two halves of the pseudo-symmetric operator site are significantly different. The two halves of the complex are most similar near the outer edge of the operator site (in a region where the lambda and 434 repressors make similar contacts), but they become increasingly different toward the center of the operator. There are striking differences near the center of the site where it appears that the arm makes significant contacts to only one half of the DNA site. This suggested a new way of aligning the operator sites in phage lambda. The high resolution structure confirms many of the previously noted features of the complex, but also reveals a number of new protein-DNA contacts. It also gives a better view of the extensive H-bonding networks that couple contacts made by different residues and different regions of the protein, and reveals important new details about the helix-turn-helix (HTH) region, and the positions of many water molecules in the complex.     

Refined crystal structure of the influenza virus N9 neuraminidase-NC41 Fab complex.

The crystal structure of the complex between neuraminidase from influenza virus (subtype N9 and isolated from an avian source) and the antigen-binding fragment (Fab) of monoclonal antibody NC41 has been refined by both least-squares and simulated annealing methods to an R-factor of 0.191 using 31,846 diffraction data in the resolution range 8.0 to 2.5 A. The resulting model has a root-mean-square deviation from ideal bond-length of 0.016 A. One fourth of the tetrameric complex comprises the crystallographic model, which has 6577 non-hydrogen atoms and consists of 389 protein residues and eight carbohydrate residues in the neuraminidase, 214 residues in the Fab light chain, and 221 residues in the heavy chain. One putative Ca ion buried in the neuraminidase, and 73 water molecules, are also included. A remarkable shape complementarity exists between the interacting surfaces of the antigen and the antibody, although the packing density of atoms at the interface is somewhat looser than in the interior of a protein. Similarly, there is a high degree of chemical complementarity between the antigen and antibody, mediated by one buried salt-link, two solvated salt-links and 12 hydrogen bonds. The antibody-binding site on neuraminidase is discontinuous and comprises five chain segments and 19 residues in contact, whilst 33 neuraminidase residues in eight segments have 899 A2 of surface area buried by the interaction (to a 1.7 A probe), including two hexose units. Seventeen residues in NC41 Fab lying in five of the six complementarity determining regions (CDRs) make contact with the neuraminidase and 36 antibody residues in seven segments have 916 A2 of buried surface area. The interface is more extensive than those of the three lysozyme-Fab complexes whose crystal structures have been determined, as judged by buried surface area and numbers of contact residues. There are only small differences (less than 1.5 A) between the complexed and uncomplexed neuraminidase structures and, at this resolution and accuracy, those differences are not unequivocal. The main-chain conformations of five of the CDRs follow the predicted canonical structures. The interface between the variable domains of the light and heavy chains is not as extensive as in other Fabs, due to less CDR-CDR interaction in NC41. The first CDR on the NC41 Fab light chain is positioned so that it could sterically hinder the approach of small as well as large substrates to the neuraminidase active-site pocket, suggesting a possible mechanism for the observed inhibition of enzyme activity by the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.     

Amine free crystal structure: the crystal structure of d(CGCGCG)2 and methylamine complex crystal

We succeeded in the crystallization of d(CGCGCG)2 and methylamine Complex. The crystal was clear and of sufficient size to collect the X-ray crystallographic data up to 1.0 A resolution using synchrotron radiation. As a result of X-ray crystallographic analysis of 2Fo-Fc map was much clear and easily traced. It is the first time monoamine co-crystallizes with d(CGCGCG)2. However, methylamine was not found from the complex crystal of d(CGCGCG)2 and methylamine. Five Mg ions were found around d(CGCGCG)2 molecules. These Mg ions neutralized the anion of 10 values of the phosphate group of DNA with five Mg2+. DNA stabilized only by a metallic ion and there is no example of analyzing the X-ray crystal structure like this. Mg ion stabilizes the conformation of Z-DNA. To use monoamine for crystallization of DNA, we found that we can get only d(CGCGCG)2 and Mg cation crystal. Only Mg cation can stabilize the conformation of Z-DNA. The method of using the monoamine for the crystallization of DNA can be applied to the crystallization of DNA of long chain of length in the future like this.     

Refined crystal structure of deoxyhemoglobin S. II. Molecular interactions in the crystal

The refined crystal structure of deoxyhemoglobin S (Padlan, E. A., and Love, W. E. (1985) J. Biol. Chem. 260, 8272-8279) was used to analyze in detail the molecular interactions between hemoglobin tetramers in the crystal. The analysis confirms the close similarity and also the nonequivalence of the molecular interactions involving the two independent tetramers in the asymmetric unit of the crystal. The residue at the site of the hemoglobin S mutation, beta 6, is intimately involved in the lateral contacts between adjacent molecules. The molecular contacts in the crystals of deoxyhemoglobin S, deoxyhemoglobin A, and deoxyhemoglobin F were compared; some contacts involve the same regions of the molecule although the details of the interactions are very different. The effect of introducing an R state tetramer into the deoxyhemoglobin S strands was investigated using the known structure of carbon monoxyhemoglobin A. It was found that substituting a molecule of carbon monoxyhemoglobin A for one of the deoxyhemoglobin S tetramers results in extensive molecular interpenetration.     

Refined crystal structure of ascorbate oxidase at 1.9 A resolution.

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)     

Refined crystal structure of type III chloramphenicol acetyltransferase at 1.75 A resolution

High level bacterial resistance to chloramphenicol is generally due to O-acetylation of the antibiotic in a reaction catalysed by chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) in which acetyl-coenzyme A is the acyl donor. The crystal structure of the type III enzyme from Escherichia coli with chloramphenicol bound has been determined and refined at 1.75 A resolution, using a restrained parameter reciprocal space least squares procedure. The refined model, which includes chloramphenicol, 204 solvent molecules and two cobalt ions has a crystallographic R-factor of 18.3% for 27,300 reflections between 6 and 1.75 A resolution. The root-mean-square deviation in bond lengths from ideal values is 0.02 A. The cobalt ions play a crucial role in stabilizing the packing of the molecule in the crystal lattice. CAT is a trimer of identical subunits (monomer Mr 25,000) and the trimeric structure is stabilized by a number of hydrogen bonds, some of which result in the extension of a beta-sheet across the subunit interface. Chloramphenicol binds in a deep pocket located at the boundary between adjacent subunits of the trimer, such that the majority of residues forming the binding pocket belong to one subunit while the catalytically essential histidine belongs to the adjacent subunit. His195 is appropriately positioned to act as a general base catalyst in the reaction, and the required tautomeric stabilization is provided by an unusual interaction with a main-chain carbonyl oxygen.     

Refined crystal structure of calcium-liganded carp parvalbumin 4.25 at 1.5-A resolution

The crystal structure of carp parvalbumin (pI = 4.25) has been refined by restrained least-squares analysis employing X-ray diffractometer data to 1.5-A resolution. The final residual for 12,653 reflections between 10 and 1.5 A with I(hkl) greater than 2 sigma(I) is 0.215. A total of 74 solvent molecules were included in the least-squares analysis. The root mean square deviation from ideality of bond lengths is 0.024 A. The model has a root mean square difference of 0.59 A from the positions of the main-chain atoms in a previously reported structure [Moews, P. C., & Kretsinger, R. H. (1975) J. Mol. Biol. 91, 201-228], which was refined by difference Fourier syntheses using data collected by film to 1.9 A. Although the overall features of the two models are very similar, there are significant differences in the amino-terminal region, which was extensively refit, and in the number of oxygen atoms liganding calcium in the CD and EF sites, which increased from six to seven in the CD site and decreased from eight to seven in the EF site.     

Refined crystal structure of ferredoxin II from Desulfovibrio gigas at 1.7 A

The crystal structure of ferredoxin II from Desulfovibrio gigas has been determined using phasing from anomalous scattering data at a resolution of 1.7 A and refined to an R-factor of 0.157. The molecule has an overall chain fold similar to that of the other bacterial ferredoxins of known structure. The molecule contains a single 3Fe-4S cluster with geometry indistinguishable from the 4Fe-4S clusters, and a disulfide bond near the site corresponding to the position of the second cluster of two-cluster ferredoxins. The cluster is bound by cysteine residues 8, 14 and 50. The side-chain of cysteine 11 extends away from the cluster, but could rotate to become the fourth cysteine ligand in the four-iron form of the molecule given a local adjustment of the polypeptide chain. This residue is modified, however, by what appears to be a methanethiol group. There are a total of eight NH . . . S bonds to the inorganic and cysteine sulfur atoms of the Fe-S cluster. There is an additional residue found that is not reported for the chemical sequence: according to the electron density a valine residue should be inserted after residue 55.     

Refined crystal structure of Cd, Zn metallothionein at 2.0 A resolution

The crystal structure of Cd5,Zn2-metallothionein from rat liver has been refined at 2.0 A resolution of a R-value of 0.176 for all observed data. The five Cd positions in the asymmetric unit of the crystal create a pseudo-centrosymmetric constellation about a crystallographic 2-fold axis. Consequently, the distribution of anomalous differences is almost ideally centrosymmetric. Therefore, the previously reported metal positions and the protein model derived therefrom are incorrect. Direct methods were applied to the protein amplitudes to locate the Cd positions. The new positions were used to calculate a new electron density map based on the Cd anomalous scattering and partial structure to model the metal clusters and the protein. Phases calculated from this model predict the positions of three sites in a (NH4)2WS4 derivative. Single isomorphous replacement phases calculated with these tungsten sites confirm the positions of the Cd sites from the new direct methods calculations. The refined metallothionein structure has a root-mean-square deviation of 0.016 A from ideality of bonds and normal stereochemistry of phi, phi and chi torsion angles. The metallothionein crystal structure is in agreement with the structures for the alpha and beta domains in solution derived by nuclear magnetic resonance methods. The overall chain folds and all metal to cysteine bonds are the same in the two structure determinations. The handedness of a short helix in the alpha-domain (residues 41 to 45) is the same in both structures. The crystal structure provides information concerning the metal cluster geometry and cysteine solvent accessibility and side-chain stereochemistry. Short cysteine peptide sequences repeated in the structure adopt restricted conformations which favor the formation of amide to sulfur hydrogen bonds. The crystal packing reveals intimate association of molecules about the diagonal 2-fold axes and trapped ions of crystallization (modeled as phosphate and sodium). Variation in the chemical and structural environments of the metal sites is in accord with data for metal exchange reactions in metallothioneins.     

Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex

Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.     

Refined crystal structure of an octanucleotide duplex with G . T mismatched base-pairs

Single crystal X-ray diffraction techniques have been used to determine the structure of the DNA octamer d(G-G-G-G-C-T-C-C) at a resolution of 2.25 A. The asymmetric unit consists of two strands coiled about each other to produce an A-type DNA helix. The double helix contains six G . C Watson-Crick base-pairs and two G . T mismatched base-pairs. The mismatches adopt a "wobble" type structure in which both bases retain their major tautomer forms. The double helix is able to accommodate this G . T pairing with little distortion of the overall helical conformation. Crystals of this octamer melt at a substantially lower temperature than do those of a related octamer also containing two G . T base-pairs. We attribute this destabilization to disruption of the hydration network around the mismatch site combined with changes in intermolecular packing. Full details are given of conformational parameters, base stacking, intermolecular contacts and hydration involving 52 solvent molecules.     

Conformational Changes in the tryptophan synthase from a hyperthermophile upon alpha2beta2 complex formation: crystal structure of the complex

The three-dimensional structure of the bifunctional tryptophan synthase alpha(2)beta(2) complex from Pyrococcus furiosus was determined by crystallographic analysis. This crystal structure, with the structures of an alpha subunit monomer and a beta(2) subunit dimer that have already been reported, is the first structural set in which changes in structure that occur upon the association of the individual tryptophan synthase subunits were observed. To elucidate the structural basis of the stimulation of the enzymatic activity of each of the alpha and beta(2) subunits upon alpha(2)beta(2) complex formation, the conformational changes due to complex formation were analyzed in detail compared with the structures of the alpha monomer and beta(2) subunit dimer. The major conformational changes due to complex formation occurred in the region correlated with the catalytic function of the enzyme as follows. (1) Structural changes in the beta subunit were greater than those in the alpha subunit. (2) Large movements of A46 and L165 in the alpha subunit due to complex formation caused a more open conformation favoring the entry of the substrate at the alpha active site. (3) The major changes in the beta subunit were the broadening of a long tunnel through which the alpha subunit product (indole) is transferred to the beta active site and the opening of an entrance at the beta active site. (4) The changes in the conformations of both the alpha and beta subunits due to complex formation contributed to the stabilization of the subunit association, which is critical for the stimulation of the enzymatic activities.     

The crystal structure of the Escherichia coli maltodextrin phosphorylase-acarbose complex

Acarbose is a naturally occurring pseudo-tetrasaccharide. It has been used in conjunction with other drugs in the treatment of diabetes where it acts as an inhibitor of intestinal glucosidases. To probe the interactions of acarbose with other carbohydrate recognition enzymes, the crystal structure of E. coli maltodextrin phosphorylase (MalP) complexed with acarbose has been determined at 2.95 A resolution and refined to crystallographic R-values of R (Rfree) = 0.241 (0.293), respectively. Acarbose adopts a conformation that is close to its major minimum free energy conformation in the MalP-acarbose structure. The acarviosine moiety of acarbose occupies sub-sites +1 and +2 and the disaccharide sub-sites +3 and +4. (The site of phosphorolysis is between sub-sites -1 and +1.) This is the first identification of sub-sites +3 and +4 of MalP. Interactions of the glucosyl residues in sub-sites +2 and +4 are dominated by carbohydrate stacking interactions with tyrosine residues. These tyrosines (Tyr280 and Tyr613, respectively, in the rabbit muscle phosphorylase numbering scheme) are conserved in all species of phosphorylase. A glycerol molecule from the cryoprotectant occupies sub-site -1. The identification of four oligosaccharide sub-sites, that extend from the interior of the phosphorylase close to the catalytic site to the exterior surface of MalP, provides a structural rationalization of the substrate selectivity of MalP for a pentasaccharide substrate. Crystallographic binding studies of acarbose with amylases, glucoamylases, and glycosyltranferases and NMR studies of acarbose in solution have shown that acarbose can adopt two different conformations. This flexibility allows acarbose to target a number of different enzymes. The two alternative conformations of acarbose when bound to different carbohydrate enzymes are discussed.     

Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin.

The crystal structure of the complex formed between eglin c, an elastase inhibitor from the medical leech, and subtilisin Carlsberg has been determined at 1.2 A resolution by a combination of Patterson search methods and isomorphous replacement techniques. The structure has been refined to a crystallographic R-value of 0.18 (8-1.2 A). Eglin consists of a four-stranded beta-sheet with an alpha-helical segment and the protease-binding loop fixed on opposite sides. This loop, which contains the reactive site Leu45I--Asp46I, is mainly held in its conformation by unique electrostatic/hydrogen bond interactions of Thr44I and Asp46I with the side chains of Arg53I and Arg51I which protrude from the hydrophobic core of the molecule. The conformation around the reactive site is similar to that found in other proteinase inhibitors. The nine residues of the binding loop Gly40I--Arg48I are involved in direct contacts with subtilisin. In this interaction, eglin segment Pro42I--Thr44I forms a three-stranded anti-parallel beta-sheet with subtilisin segments Gly100--Gly102 and Ser125--Gly127. The reactive site peptide bond of eglin is intact, and Ser221 OG of the enzyme is 2.81 A apart from the carbonyl carbon.     

Attenuating lymphocyte activity: the crystal structure of the BTLA-HVEM complex

Five CD28-like proteins exert positive or negative effects on immune cells. Only four of these five receptors interact with members of the B7 family. The exception is BTLA (B and T lymphocyte attenuator), which instead interacts with the tumor necrosis factor receptor superfamily member HVEM (herpes virus entry mediator). To better understand this interaction, we determined the 2.8-A crystal structure of the BTLA-HVEM complex. This structure shows that BTLA binds the N-terminal cysteine-rich domain of HVEM and employs a unique binding surface compared with other CD28-like receptors. Moreover, the structure shows that BTLA recognizes the same surface on HVEM as gD (herpes virus glycoprotein D) and utilizes a similar binding motif. Light scattering analysis demonstrates that the extracellular domain of BTLA is monomeric and that BTLA and HVEM form a 1:1 complex. Alanine-scanning mutagenesis of HVEM was used to further define critical binding residues. Finally, BTLA adopts an immunoglobulin I-set fold. Despite structural similarities to other CD28-like members, BTLA represents a unique co-receptor.     

Refined crystal structure of troponin C from turkey skeletal muscle at 2.0 A resolution

The crystal structure of troponin C from turkey skeletal muscle has been refined at 2.0 A resolution (1 A = 0.1 nm). The resulting crystallographic R factor (R = sigma[[Fo[-[Fc[[/sigma[Fo[, where [Fo[ and [Fc[ are the observed and calculated structure factor amplitudes) is 0.155 for the 8054 reflections with intensities I greater than or equal to 2 sigma(I) within the 10 A to 2.0 A resolution range. With 66% of the residues in helical conformation, troponin C provides a good sample for helix analysis. The mean alpha-helix dihedral angles (phi, psi = -62 degrees, -42 degrees) agree with values observed for helical regions in other proteins. The helices are all curved and/or kinked. In particular, the 31 amino acid long inter-domain helix is smoothly curved, with a rather large radius of curvature of 137 A. Helix packing is different in the Ca2+-free domain (N-terminal) and the Ca2+-bound domain (C-terminal). The inter-helix angles for the two helix-loop-helix motifs in the regulatory domain are 133 degrees and 151 degrees, whereas the value for the two motifs in the C-terminal domain is 110 degrees, as observed in the EF-hands of parvalbumin. These differences affect the packing of the respective hydrophobic cores of each domain, in particular the disposition of aromatic rings. Pairwise arrangement of Ca2+-binding loops is common to both states, but the conformation is markedly different. Conversion of one to the other can be achieved by small cumulative changes of main-chain dihedral angles. The integrity of loop structure is maintained by numerous electrostatic interactions. Both salt bridges and carboxyl-carboxylate interactions are observed in TnC. There are more intramolecular (9) than intermolecular (1) salt bridges. Carboxyl-carboxylate interactions occur because the pH of the crystals is 5.0 and there is a multitude of aspartate and glutamate residues. One is intramolecular and four are intermolecular. Polar side-chain interactions occur more commonly with main-chain carbonyls and amides than with other polar side-chains. These interactions are mostly short range, and are similar to those observed in other proteins with one exception: negatively charged side-chains interact more frequently with main-chain carbonyl oxygen atoms. However, out of 19 such interactions, 10 involve oxygen atoms of the Ca2+ ligands. These unfavorable interactions are compensated by the favorable interactions with the Ca2+ ions and with main-chain amides. They are a trivial consequence of the tight fold of the Ca2+-binding loops.     

Refined crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.0 A resolution

The crystal structure of a class A beta-lactamase from Staphylococcus aureus PC1 has been refined at 2.0 A resolution. The resulting crystallographic R-factor (R = sigma h parallel Fo[-]Fc parallel/sigma h[Fo], where [Fo] and [Fc] are the observed and calculated structure factor amplitudes, respectively), is 0.163 for the 17,547 reflections with I greater than or equal to 2 sigma (I) within the 8.0 A to 2.0 A resolution range. The molecule consists of two closely associated domains. One domain is formed by a five-stranded antiparallel beta-sheet with three helices packing against a face of the sheet. The second domain is formed mostly by helices that pack against the second face of the sheet. The active site is located in the interface between the two domains, and many of the residues that form it are conserved in all known sequences of class A beta-lactamases. Similar to the serine proteases, an oxyanion hole is implicated in catalysis. It is formed by two main-chain nitrogen atoms, that of the catalytic seryl residue, Ser70, and that of Gln237 on an edge beta-strand of the major beta-sheet. Ser70 is interacting with another conserved seryl residue, Ser130, located between the two ammonium groups of the functionally important lysine residues, Lys73 and Lys234. Such intricate interactions point to a possible catalytic role for this second seryl residue. Another key catalytic residue is Glu166. There are several unusual structural features associated with the active site. (1) A cis peptide bond has been identified between the catalytic Glu166 and Ile167. (2) Ala69 and Leu220 have strained phi, psi dihedral angles making close contacts that restrict the conformation of the active site beta-strand involved in the formation of the oxyanion hole. (3) A buried aspartate residue, the conserved Asp233, is located next to the active site Lys234. It is interacting with another buried aspartyl residue, Asp246. An internal solvent molecule is also involved, but the rest of its interactions with the protein indicate it is not a cation. (4) Another conserved aspartyl residue that is desolvated is Asp131, adjacent to Ser130. Its charge is stabilized by interactions with four main-chain nitrogen atoms. (5) An internal cavity underneath the active site depression is filled with six solvent molecules. This, and an adjacent cavity occupied by three solvent molecules partially separate the omega-loop associated with the active site from the rest of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphoprotein-protein interactions revealed by the crystal structure of kinase-associated phosphatase in complex with phosphoCDK2.

The CDK-interacting protein phosphatase KAP dephosphorylates phosphoThr-160 (pThr-160) of the CDK2 activation segment, the site of regulatory phosphorylation that is essential for kinase activity. Here we describe the crystal structure of KAP in association with pThr-160-CDK2, representing an example of a protein phosphatase in complex with its intact protein substrate. The major protein interface between the two molecules is formed by the C-terminal lobe of CDK2 and the C-terminal helix of KAP, regions remote from the kinase-activation segment and the KAP catalytic site. The kinase-activation segment interacts with the catalytic site of KAP almost entirely via the phosphate group of pThr-160. This interaction requires that the activation segment is unfolded and drawn away from the kinase molecule, inducing a conformation of CDK2 similar to the activated state observed in the CDK2/cyclin A complex.