Functional and structural characterisation of RimL from Bacillus cereus, a new Nα-acetyltransferase of ribosomal proteins that was wrongly assigned as an aminoglycosyltransferase.

Enzymes of the GNAT (GCN5-relate N-acetyltransferases) superfamily are important regulators of cell growth and development. They are functionally diverse and share low amino acid sequence identity, making functional annotation difficult. In this study, we report the function and structure of a new ribosomal enzyme, Nα-acetyl transferase from Bacillus cereus (RimLBC), a protein that was previously wrongly annotated as an aminoglycosyltransferase. Firstly, extensive comparative amino acid sequence analyses suggested RimLBC belongs to a cluster of proteins mediating acetylation of the ribosomal protein L7/L12. To assess if this was the case, several well established substrates of aminoglycosyltransferases were screened. The results of these studies did not support an aminoglycoside acetylating function for RimLBC. To gain further insight into RimLBC biological role, a series of studies that included MALDI-TOF, isothermal titration calorimetry, NMR, X-ray protein crystallography, and site-directed mutagenesis confirmed RimLBC affinity for Acetyl-CoA and that the ribosomal protein L7/L12 is a substrate of RimLBC. Last, we advance a mechanistic model of RimLBC mode of recognition of its protein substrates. Taken together, our studies confirmed RimLBC as a new ribosomal Nα-acetyltransferase and provide structural and functional insights into substrate recognition by Nα-acetyltransferases and protein acetylation in bacteria.

Exploring the chemical space of the lysine-binding pocket of the first kringle domain of hepatocyte growth factor/scatter factor (HGF/SF) yields a new class of inhibitors of HGF/SF-MET binding.

The growth/motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, constitute a signalling system essential for embryogenesis and for tissue/organ regeneration in post-natal life. HGF/SF-MET signalling, however, also plays a key role in the onset of metastasis of a large number of human tumours. Both HGF/SF and MET are high molecular weight proteins that bury an extensive interface upon complex formation and thus constitute a challenging target for the development of low molecular weight inhibitors. Here we have used surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and X-ray crystallography to screen a diverse fragment library of 1338 members as well as a range of piperazine-like compounds. Several small molecules were found to bind in the lysine-binding pocket of the kringle 1 domain of HGF/SF and its truncated splice variant NK1. We have defined the binding mode of these compounds, explored their biological activity and we show that selected fragments inhibit MET downstream signalling. Thus we demonstrate that targeting the lysine-binding pocket of NK1 is an effective strategy to generate MET receptor antagonists and we offer proof of concept that the HGF/SF-MET interface may be successfully targeted with small molecules. These studies have broad implications for the development of HGF/SF-MET therapeutics and cancer treatment.

An analysis of subdomain orientation, conformational change and disorder in relation to crystal packing of aspartic proteinases.

The analysis reported here describes detailed structural studies of endothiapepsin (the aspartic proteinase from Endothia parasitica), with and without bound inhibitors, and human pepsin 3b. Comparison of multiple crystal structures of members of the aspartic proteinase family has revealed small but significant differences in domain orientation in different crystal forms. In this paper, it is shown that these differences in domain orientation do not necessarily correlate with the presence or absence of bound inhibitors, but appear to stem at least partly from crystal contacts mediated by sulfate ions. However, since the same inherent flexibility of the structure is observed for other enzymes in this family such as human pepsin, the native structure of which is also reported here, the observed domain movements may well have implications for the mechanism of catalysis.

FSH and TSH binding to their respective receptors: similarities, differences and implication for glycoprotein hormone specificity.

The crystal structures of the leucine-rich repeat domain (LRD) of the FSH receptor (FSHR) in complex with FSH and the TSH receptor (TSHR) LRD in complex with the thyroid-stimulating autoantibody (M22) provide opportunities to assess the molecular basis of the specificity of glycoprotein hormone-receptor binding. A comparative model of the TSH-TSHR complex was built using the two solved crystal structures and verified using studies on receptor affinity and activation. Analysis of the FSH-FSHR and TSH-TSHR complexes allowed identification of receptor residues that may be important in hormone-binding specificity. These residues are in leucine-rich repeats at the two ends of the FSHR and the TSHR LRD structures but not in their central repeats. Interactions in the interfaces are consistent with a higher FSH-binding affinity for the FSHR compared with the binding affinity of TSH for the TSHR. The higher binding affinity of porcine (p)TSH and bovine (b)TSH for human (h)TSHR compared with hTSH appears not to be dependent on interactions with the TSHR LRD as none of the residues that differ among hTSH, pTSH or bTSH interact with the LRD. This suggests that TSHs are likely to interact with other parts of the receptors in addition to the LRD with these non-LRD interactions being responsible for affinity differences. Analysis of interactions in the FSH-FSHR and TSH-TSHR complexes suggests that the alpha-chains of both hormones tend to be involved in the receptor activation process while the beta-chains are more involved in defining binding specificity.

Quantifying structural and functional restraints on amino acid substitutions in evolution of proteins.

One of Oleg Ptitsyn's most important papers (Shakhnovich, E., Abkevich, V., and Ptitsyn, O. (1996) Nature, 379, 96-98) describes how knowledge of structure and function can be used to understand better the nature of amino acid substitutions in families and superfamilies of proteins. The selective advantages of retaining structure and function during evolution can be expressed as restraints on the amino acid substitutions that are accepted.

Characteristics of a monoclonal antibody to the thyrotropin receptor that acts as a powerful thyroid-stimulating autoantibody antagonist.

Analysis of nine mouse monoclonal antibodies (mAbs) to the thyrotropin receptor (TSHR) with TSH antagonist activity showed that only one of the mAbs (RSR B2) was an effective antagonist of the human thyroid stimulating autoantibody M22. Crystals of B2 Fab were analyzed by x-ray diffraction and a crystal structure at 3.3 A resolution was obtained. The surface charge and topography of the B2 antigen binding site were markedly different from those of the thyroid-stimulating mAb M22 and these differences might contribute to the different properties of the two mAbs. B2 (but not other mouse TSHR-specific mAbs) was also an effective antagonist of thyroid stimulating autoantibody activity in 14 of 14 different sera from patients with Graves' disease. 125I-labeled B2 bound to the TSHR with high affinity (2 x 10(10) L/mol) and patient serum TSHR autoantibodies inhibited labeled B2 binding to the receptor in a similar way to inhibition of labeled TSH binding (r = 0.75; n = 20). Furthermore, labeled B2 binding was inhibited by patient serum TSHR autoantibodies with TSH antagonist activity and also by mouse and human thyroid stimulating mAbs. Overall, mAb B2 is a powerful antagonist of thyroid stimulating autoantibodies (and TSH) thus resembling closely patient serum TSH antagonist TSHR autoantibodies. Furthermore, B2 might have potentially important in vivo applications when tissues containing the TSHR (including those in the orbit) need to be made unresponsive to stimulating autoantibodies.

The conserved N-terminal region of the mitotic checkpoint protein BUBR1: a putative TPR motif of high surface activity.

BUBR1, a key component of the mitotic spindle checkpoint, is a multidomain protein kinase that is activated in response to kinetochore tension. Although BUB1 and BUBR1 play an important role in cell division, very little is known about their structural characteristics. We show that the conserved N-terminal region of BUBR1, comprising residues 1-204, is a globular domain of high alpha-helical content ( approximately 60%), stable in the pH range 4-9 and probably organized as a tetratricopeptide motif repeat (TPR), most closely resembling residues 16-181 of protein phosphatase 5. Because the latter presents a continuous amphipathic groove and is regulated by binding certain fatty acids, we compared the properties of BUBR1(1-204) and TPR-PP5(16-181) at air/water interfaces and found that both proteins exhibited a similar surface activity and formed stable, rigid monolayers. The deletion of a region that probably comprises several alpha-helices of BUBR1 indicates that long-range interactions are essential for the stability of the N-terminal domain. The presence of the putative TPR motif strongly suggests that the N-terminal domain of BUBR1 is involved in direct protein-protein interactions and/or protein-lipid interactions.

Pantothenate biosynthesis in higher plants.

Pantothenate (vitamin B5) is a water-soluble vitamin essential for the synthesis of CoA and ACP (acyl-carrier protein, cofactors in energy yielding reactions including carbohydrate metabolism and fatty acid synthesis. Pantothenate is synthesized de novo by plants and micro-organisms; however, animals obtain the vitamin through their diet. Utilizing our knowledge of the pathway in Escherichia coli, we have discovered and cloned genes encoding the first and last enzymes of the pathway from Arabidopsis, panB1, panB2 and panC. It is unlikely that there is a homologue of the E. coli panD gene, therefore plants must make beta-alanine by an alternative route. Possible candidates for the remaining gene, panE, are being investigated. GFP (green fluorescent protein) fusions of the three identified plant enzymes have been generated and the subcellular localization of the enzymes studied. Work is now being performed to elucidate expression patterns of the transcripts and characterize the proteins encoded by these genes.

Properties of polyproline II, a secondary structure element implicated in protein-protein interactions.

The polyproline II (PPII) conformation of protein backbone is an important secondary structure type. It is unusual in that, due to steric constraints, its main-chain hydrogen-bond donors and acceptors cannot easily be satisfied. It is unable to make local hydrogen bonds, in a manner similar to that of alpha-helices, and it cannot easily satisfy the hydrogen-bonding potential of neighboring residues in polyproline conformation in a manner analogous to beta-strands. Here we describe an analysis of polyproline conformations using the HOMSTRAD database of structurally aligned proteins. This allows us not only to determine amino acid propensities from a much larger database than previously but also to investigate conservation of amino acids in polyproline conformations, and the conservation of the conformation itself. Although proline is common in polyproline helices, helices without proline represent 46% of the total. No other amino acid appears to be greatly preferred; glycine and aromatic amino acids have low propensities for PPII. Accordingly, the hydrogen-bonding potential of PPII main-chain is mainly satisfied by water molecules and by other parts of the main-chain. Side-chain to main-chain interactions are mostly nonlocal. Interestingly, the increased number of nonsatisfied H-bond donors and acceptors (as compared with alpha-helices and beta-strands) makes PPII conformers well suited to take part in protein-protein interactions.

Characteristics of a human monoclonal autoantibody to the thyrotropin receptor: sequence structure and function.

The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described. Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation. Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation. Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22. These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity. Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation. The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain. The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction.

Crystallization and X-ray analysis of the Y75N mutant of Mucor pusillus pepsin complexed with inhibitor.

Y75N mutant Mucor pusillus pepsin has been overexpressed in yeast, purified and cocrystallized with the iodine-containing human renin inhibitor CP-113972 [(2R,3S]-isopropyl 3-[(L-prolyl-p-iodo-L-phenylalanyl-S-methyl-cysteinyl)amino-4]-cyclohexyl-2-hydroxybutanoate] for X-ray crystallography. Tetragonal complex crystals with space group P4(3)2(1)2 were produced by the hanging-drop vapour-diffusion method and diffracted to 3.0 A. The crystals exhibited unit-cell parameters a = b = 182.5, c = 99.1 A and contained four molecules in the asymmetric unit. A 96% complete data set was collected at 298 K using Cu Kalpha X-rays from a rotating-anode generator. Solution of the crystal structure of Y75N mutant M. pusillus pepsin is under way by molecular replacement using the molecular coordinates of wild-type M. pusillus pepsin as a model.

DDBASE2.0: updated domain database with improved identification of structural domains.

MOTIVATION: Although many methods are available for the identification of structural domains from protein three-dimensional structures, accurate definition of protein domains and the curation of such data for a large number of proteins are often possible only after manual intervention. The availability of domain definitions for protein structural entries is useful for the sequence analysis of aligned domains, structure comparison, fold recognition procedures and understanding protein folding, domain stability and flexibility. RESULTS: We have improved our method of domain identification starting from the concept of clustering secondary structural elements, but with an intention of reducing the number of discontinuous segments in identified domains. The results of our modified and automatic approach have been compared with the domain definitions from other databases. On a test data set of 55 proteins, this method acquires high agreement (88%) in the number of domains with the crystallographers' definition and resources such as SCOP, CATH, DALI, 3Dee and PDP databases. This method also obtains 98% overlap score with the other resources in the definition of domain boundaries of the 55 proteins. We have examined the domain arrangements of 4592 non-redundant protein chains using the improved method to include 5409 domains leading to an update of the structural domain database. AVAILABILITY: The latest version of the domain database and online domain identification methods are available from http://www.ncbs.res.in/~faculty/mini/ddbase/ddbase.html SUPPLEMENTARY INFORMATION: http://www.ncbs.res.in/~faculty/mini/ddbase/supplementary/supplementary.html

Structural insights into the evolution of the pantothenate-biosynthesis pathway.

Pantothenate is synthesized in bacteria, fungi and plants, and as vitamin B5 is a dietary requirement in animals. The three-dimensional structures of the four Escherichia coli enzymes involved in the production of pantothenate have been determined. We describe the use of comparative analyses of the sequences and structures to identify distant homologues of the four enzymes in an attempt to understand the evolution of the pathway. We conclude that it is likely to have evolved via a patchwork mechanism, whereby the individual enzymes were recruited separately.

Crystallization and preliminary X-ray study of recombinant betaine-homocysteine S-methyltransferase from rat liver.

Betaine-homocysteine S-methyltransferase is one of the three enzymes involved in homocysteine catabolism. It uses betaine as the methyl donor to convert homocysteine into methionine, also producing dimethylglycine. Recombinant BHMT from rat liver was crystallized by the vapour-diffusion method in both native and seleniomethionyl-labelled forms. Crystals belong to space group P2(1), with unit-cell parameters a = 57.8, b = 149.3, c = 96.2 A, beta = 92.9 degrees. Data from native, seleniomethionine-labelled and two heavy-atom derivatives were collected using synchrotron sources. Self-rotation function and sedimentation-velocity experiments suggest that the enzyme is tetrameric with 222 symmetry.

Crystal structure of an enzyme displaying both inositol-polyphosphate-1-phosphatase and 3'-phosphoadenosine-5'-phosphate phosphatase activities: a novel target of lithium therapy.

Lithium cations exert profound and selective psychopharmacological effects on ameliorate manic-depressive psychosis. Although lithium is an effective drug for both treatment and prophylaxis of bipolar disorder, the precise mechanism of action is not well understood. Lithium acts as both an uncompetitive and non-competitive inhibitor of several lithium- sensitive phosphatases with regard to substrate and magnesium cofactor, respectively. In this work, we report the crystal structure and reaction mechanism of Rattus norvegicus 3'-phosphoadenosine 5'-phosphate and inositol 1,4-bisphosphate phosphatase (RnPIP), a recently identified target of lithium therapy. This Li(+)-sensitive enzyme plays a crucial role in several cellular processes, such as RNA processing, sulphation reactions and probably inositol recycling. RnPIP specifically removes the 3'-phosphate group of 3'-phosphoadenosine 5'-phosphate (PAP) and the 1'-phosphate group of inositol 1,4-bisphosphate (I(1),(4)P(2)) producing AMP and inositol 4'-phosphate, respectively. The crystal structure of RnPIP complexed with AMP, Pi and magnesium ions at 1.69 A resolution provides insight into the reaction mechanism of the hydrolysis of PAP. The core fold of the enzyme is equivalent to that found in other Li(+)-sensitive phosphatases, such as inositol monophosphatase, but molecular modelling of I(1),(4)P(2) in the RnPIP active site reveals important structural determinants that accommodate this additional substrate. RnPIP is potently inhibited by lithium and, as the accumulation of PAP inhibits a variety of proteins, including sulphotransferases and RNA processing enzymes, this dual specificity enzyme represents a potential target of lithium action, in addition to inositol monophosphatases.

Crystal structure of Escherichia coli ketopantoate reductase at 1.7 A resolution and insight into the enzyme mechanism.

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate on the pantothenate (vitamin B(5)) biosynthetic pathway. The Escherichia coli panE gene encoding KPR was cloned and expressed at high levels as the native and selenomethionine-substituted (SeMet) proteins. Both native and SeMet recombinant proteins were purified by three chromatographic steps, to yield pure proteins. The wild-type enzyme was found to have a K(M)(NADPH) of 20 microM, a K(M)(ketopantoate) of 60 microM, and a k(cat) of 40 s(-1). Regular prismatic KPR crystals were prepared using the hanging drop technique. They belonged to the tetragonal space group P4(2)2(1)2, with cell parameters: a = b = 103.7 A and c = 55.7 A, accommodating one enzyme molecule per asymmetric unit. The structure of KPR was determined by the multiwavelength anomalous dispersion method using the SeMet protein, for which data were collected to 2.3 A resolution. The native data were collected to 1.7 A resolution and used to refine the final structure. The secondary structure comprises 12 alpha-helices, three 3(10)-helices, and 11 beta-strands. The enzyme is monomeric and has two domains separated by a cleft. The N-terminal domain has an alphabeta-fold of the Rossmann type. The C-terminal domain (residues 170-291) is composed of eight alpha-helices. KPR is shown to be a member of the 6-phosphogluconate dehydrogenase C-terminal domain-like superfamily. A model for the ternary enzyme-NADPH-ketopantoate ternary complex provides a rationale for kinetic data reported for specific site-directed mutants.

Crystal structure of an Xrcc4-DNA ligase IV complex.

A complex of two proteins, Xrcc4 and DNA ligase IV, plays a fundamental role in DNA non-homologous end joining (NHEJ), a cellular function required for double-strand break repair and V(D)J recombination. Here we report the crystal structure of human Xrcc4 bound to a polypeptide that corresponds to the DNA ligase IV sequence linking its two BRCA1 C-terminal (BRCT) domains. In the complex, a single ligase chain binds asymmetrically to an Xrcc4 dimer. The helical tails of Xrcc4 undergo a substantial conformational change relative to the uncomplexed protein, forming a coiled coil that unwinds upon ligase binding, leading to a flat interaction surface. A buried network of charged hydrogen bonds surrounded by extensive hydrophobic contacts explains the observed tightness of the interaction. The strong conservation of residues at the interface between the two proteins provides evidence that the observed mode of interaction has been maintained in NHEJ throughout evolution.

Crystal structures of NK1-heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists of the MET receptor.

NK1 is a splice variant of the polypeptide growth factor HGF/SF, which consists of the N-terminal (N) and first kringle (K) domain and requires heparan sulfate or soluble heparin for activity. We describe two X-ray crystal structures of NK1-heparin complexes that define a heparin-binding site in the N domain, in which a major role is played by R73, with further contributions from main chain atoms of T61, K63 and G79 and the side chains of K60, T61, R76, K62 and K58. Mutagenesis experiments demonstrate that heparin binding to this site is essential for dimerization in solution and biological activity of NK1. Heparin also comes into contact with a patch of positively charged residues (K132, R134, K170 and R181) in the K domain. Mutation of these residues yields NK1 variants with increased biological activity. Thus, we uncover a complex role for heparan sulfate in which binding to the primary site in the N domain is essential for biological activity whereas binding to the K domain reduces activity. We exploit the interaction between heparin and the K domain site in order to engineer NK1 as a potent receptor agonist and suggest that dual (positive and negative) control may be a general mechanism of heparan sulfate-dependent regulation of growth factor activity.

A novel superfamily of enzymes that catalyze the modification of guanidino groups.

Three enzymes, peptidyl-arginine deiminase from Porphyromonas gingivalis, arginine deiminase and amidinotransferase are traditionally classified separately. By combining PSI-BLAST and FUGUE, data presented in this article describe how these enzymes belong to a novel superfamily, adopting a common fold and sharing similar catalytic mechanisms.

HOMSTRAD: adding sequence information to structure-based alignments of homologous protein families.

summary: We describe an extension to the Homologous Structure Alignment Database (HOMSTRAD; Mizuguchi et al., Protein Sci., 7, 2469-2471, 1998a) to include homologous sequences derived from the protein families database Pfam (Bateman et al., Nucleic Acids Res., 28, 263-266, 2000). HOMSTRAD is integrated with the server FUGUE (Shi et al., submitted, 2001) for recognition and alignment of homologues, benefitting from the combination of abundant sequence information and accurate structure-based alignments. AVAILABILITY The HOMSTRAD database is available at: http://www-cryst.bioc.cam.ac.uk/homstrad/. Query sequences can be submitted to the homology recognition/alignment server FUGUE at: http://www-cryst.bioc.cam.ac.uk/fugue/.