Biomedical subjects

M Helmer-Citterich

Publications and source records attributed to M Helmer-Citterich.

17 recordsLinked to original sources

PubMed2000

Three-dimensional view of the surface motif associated with the P-loop structure: cis and trans cases of convergent evolution.

Here we identify the determinants of the nucleotide-binding ability associated with the P-loop-containing proteins, inferring their functional importance from their structural convergence to a unique three- dimensional (3D) motif. (1) A new surface 3D pattern is identified for the P-loop nucleotide-binding region, which is more selective than the corresponding sequence pattern; (2) the signature displays one residue that we propose is the determinant for the guanine-binding ability (the residues aligned to ras D119; this residue is known to be important only in the G-proteins, we extend the prediction to all the other P-loop- containing proteins); and (3) two cases of convergent evolution at the molecular level are highlighted in the analysis of the active site: the positive charge aligned to ras K117 and the arginine residues aligned to the GAP arginine finger. The analysis of the residues conserved on protein surfaces allows one to identify new functional or evolutionary relationships among protein structures that would not be detectable by conventional sequence or structure comparison methods.

Adenosine Triphosphate↗

PubMed2000

SH3-SPOT: an algorithm to predict preferred ligands to different members of the SH3 gene family.

We have developed a procedure to predict the peptide binding specificity of an SH3 domain from its sequence. The procedure utilizes information extracted from position-specific contacts derived from six SH3/peptide or SH3/protein complexes of known structure. The framework of SH3/peptide contacts defined on the structure of the complexes is used to build a residue-residue interaction database derived from ligands obtained by panning peptide libraries displayed on filamentous phage. The SH3-specific interaction database is a multidimensional array containing frequencies of position-specific contacts. As input, SH3-SPOT requires the sequence of an SH3 domain and of a query decapeptide ligand. The array, that we call the SH3-specific matrix, is then used to evaluate the probability that the peptide would bind the given SH3 domain. This procedure is fast enough to be applied to the entire protein sequence database. Panning experiments were performed to search putative specific ligands of different SH3 domains in a database of decapeptides, or in a database of protein sequences. The procedure ranked some of the natural partners of interaction of a number of SH3 domains among the best ligands of the approximately 5. 6x10(9) different decapeptides in the SWISSPROT database. We expect the predictive power of the method to increase with the enrichment of the SH3-specific matrix by interaction data derived from new complex structures or from the characterization of new ligands. The procedure was developed using the SH3 domain family as test case but its application can easily be extended to other families of protein domains (such as, SH2, MHC, EH, PDZ, etc.).

Algorithms↗

PubMed2000

Protein surface similarities: a survey of methods to describe and compare protein surfaces.

Many methods have been developed to analyse protein sequences and structures, although less work has been undertaken describing and comparing protein surfaces. Evolution can lead sequences to diverge or structures to change topology; nevertheless, surface determinants that are essential to protein function itself may be mantained. Moreover, different molecules could converge to similar functions by gaining specific surface determinants. In such cases, sequence or structure comparisons are likely to be inadequate in describing or identifying protein functions and evolutionary relationships among proteins. Surface analysis can identify function determinants that are independent of sequence or secondary structure and can therefore be a powerful tool to highlight cases of possible convergent or divergent evolution. This kind of approach can be useful for a better understanding of protein molecular and biochemical mechanisms of catalysis or interaction with a ligand, which are usually surface dependent. Protein surface comparison, when compared to sequence or structure comparison methods, is a hard computational challenge and evaluated methods allowing the comparison of protein surfaces are difficult to find. In this review, we will survey the current knowledge about protein surface similarity and the techniques to detect it.

Algorithms↗

PubMed2000

Identification of a putative binding site for negatively charged surfaces in the fibronectin type II domain of human factor XII--an immunochemical and homology modeling approach.

Monoclonal antibodies directed against functional sites of proteins provide useful tools for structure-function studies. Here we describe a mAb, KOK5, directed against the heavy chain region of human coagulation factor XII (FXII), which inhibits kaolin-induced clotting activity by preventing the binding of FXII to kaolin. Furthermore, mAb KOK5 enhances FXII susceptibility for cleavage by kallikrein and supports FXII autoactivation. Hence, mAb KOK5 likely is directed against the binding site of FXII for negatively charged surfaces. Screening of two phage-displayed random peptide libraries with mAb KOK5 selected phages that could be grouped on the basis of two amino acid consensus sequences: A) FXFQTPXW and B) HQ/LCTHR/KKC. Sequence A contains two motifs: one shares homology with FXII amino acid residues 30-33 (FPFQ), the second one with residues 57-60 (TPNF); both amino acid stretches belonging to the fibronectin type II domain of FXII. Sequence B also reveals homology with part of the fibronectin type II domain, i.e. the stretch 40-47 (HKCTHKGR). A three-dimensional model of FXII residues 28-65, obtained by homology modeling, indicated that the three amino acid stretches 30-33, 40-47 and 57-60 are close to each other and accessible for the solvent, i.e. in a form available for interaction with the monoclonal antibody, suggesting that mAb KOK5 recognizes a discontinuous epitope on the fibronectin type III domain of FXII. Peptides corresponding to FXII sequences 29-37 (FXII29-37) or 39-47 (FXII39-47), were synthesized and tested for the capability to inhibit FXII binding to negatively charged surfaces. Peptide FXII39-47 inhibited the binding of labeled FXII to kaolin and effectively prevented both dextran sulfate- and kaolin-induced activation of the contact system in plasma. Hence, we suggest that the fibronectin type II domain of FXII, in particular residues 39 to 47, contribute to the binding site of FXII for negatively charged surfaces.

Amino Acid Sequence↗

PubMed1998

Three-dimensional profiles: a new tool to identify protein surface similarities.

We report a procedure for the description and comparison of protein surfaces, which is based on a three-dimensional (3D) transposition of the profile method for sensitive protein homology sequence searches. Although the principle of the method can be applied to detect similarities to a single protein surface, the possibility of extending this approach to protein families displaying common structural and/or functional properties, makes it a more powerful tool. In analogy to profiles derived from the multiple alignment of protein sequences, we derive a 3D surface profile from a protein structure or from a multiple structure alignment of several proteins. The 3D profile is used to screen the protein structure database, searching for similar protein surfaces. The application of the procedure to SH2 and SH3 binding pockets and to the nucleotide binding pocket associated with the p-loop structural motif is described. The SH2 and SH3 3D profiles can identify all the SH2 and SH3 binding regions present in the test dataset; the p-loop 3D profile is able to recognize all the p-loop-containing proteins present in the test dataset. Analysis of the p-loop 3D profile allowed the identification of a positive charge whose position is conserved in space but not in sequence. The best ranking non-p-loop-containing protein is an ADP-forming succinyl coenzyme A synthetase, whose nucleotide-binding region has not yet been identified.

Amino Acid Sequence↗

PubMed1998

Design and properties of a Myc derivative that efficiently homodimerizes.

bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and specific protein-protein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and off cell proliferation, differentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization specificity. We report that Myc recognition specificity is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes efficiently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding efficiency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.

3T3 Cells↗

PubMed1998

Structural studies on an inhibitory antibody against Thermus aquaticus DNA polymerase suggest mode of inhibition.

TP7, an antibody against Thermus aquaticus DNA polymerase I (TaqP), is used as a thermolabile switch in 'hot start' variations of PCR to minimize non-specific amplification events. Earlier studies have established that TP7 binds to the polymerase domain of TaqP, competes with primer template complex for binding and is a potent inhibitor of the polymerase activity of TaqP. We report crystallographic determination of the structure of an Fab fragment of TP7 and computational docking of the structure with the known three-dimensional structure of the enzyme. Our observations strongly suggest that the origin of inhibitory ability of TP7 is its binding to enzyme residues involved in DNA binding and polymerization mechanism. Although criteria unbiased by extant biochemical data have been used in identification of a putative solution, the resulting complex offers an eminently plausible structural explanation of biochemical observations. The results presented are of general significance for interpretation of docking experiments and in design of small molecular inhibitors of TaqP, that are not structurally similar to substrates, for use in PCR. Structural and functional similarities noted among DNA polymerases, and the fact that several DNA polymerases are pharmacological targets, make discovery of non-substrate based inhibitors of additional importance.

Amino Acid Sequence↗

PubMed1997

Correlated mutations contain information about protein-protein interaction.

Many proteins have evolved to form specific molecular complexes and the specificity of this interaction is essential for their function. The network of the necessary inter-residue contacts must consequently constrain the protein sequences to some extent. In other words, the sequence of an interacting protein must reflect the consequence of this process of adaptation. It is reasonable to assume that the sequence changes accumulated during the evolution of one of the interacting proteins must be compensated by changes in the other. Here we apply a method for detecting correlated changes in multiple sequence alignments to a set of interacting protein domains and show that positions where changes occur in a correlated fashion in the two interacting molecules tend to be close to the protein-protein interfaces. This leads to the possibility of developing a method for predicting contacting pairs of residues from the sequence alone. Such a method would not need the knowledge of the structure of the interacting proteins, and hence would be both radically different and more widely applicable than traditional docking methods. We indeed demonstrate here that the information about correlated sequence changes is sufficient to single out the right inter-domain docking solution amongst many wrong alternatives of two-domain proteins. The same approach is also used here in one case (haemoglobin) where we attempt to predict the interface of two different proteins rather than two protein domains. Finally, we report here a prediction about the inter-domain contact regions of the heat- shock protein Hsc70 based only on sequence information.

Amino Acid Sequence↗

PubMed1997

Homology modeling of Neurospora crassa geranylgeranyl pyrophosphate synthase: structural interpretation of mutant phenotypes.

A model of the tertiary structure of the Neurospora crassa carotenogenic prenyltransferase, geranylgeranyl pyrophosphate synthase (GGPPS), is presented, based on structural homology with other prenyltransferases and on the crystal structure of recombinant avian farnesyl pyrophosphate synthase (FPPS). The conserved aspartate-rich motifs DDxx(xx)D and associated basic residues, considered to be the active sites for binding and catalysis in all prenyltransferases, are highly conserved in the N. crassa GGPPS protein, while other regions display a lower degree of sequence homology; thus the GGPPS model structure is predicted to be highly reliable in the active site region. A number of carotene-deficient mutants have been generated utilizing the repeat-induced point mutation (RIP) mechanism: mutant al-3RIP1 carries a Ser-to-Asn mutation in position 336 which falls within the predicted active site of the enzyme. Analysis of the model structure of this mutant indicates that Ser336 may be involved in substrate uptake. Two other mutants, al-3RIP3 and al-3RIP6, carry mutations in positions in the GGPPS protein, homologous to regions of the avian FPPS enzyme proposed to be involved in enzyme dimerization and substrate uptake, respectively, suggesting an explanation for the reduced carotene content of these mutants.

Alkyl and Aryl Transferases↗

PubMed1997

ESCHER: a new docking procedure applied to the reconstruction of protein tertiary structure.

Evaluation of Surface Complementarity, Hydrogen bonding, and Electrostatic interaction in molecular Recognition (ESCHER) is a new docking procedure consisting of three modules that work in series. The first module evaluates the geometric complementarity and produces a set of rough solutions for the docking problem. The second module identifies molecular collisions within those solutions, and the third evaluates their electrostatic complementarity. We describe the algorithm and its application to the docking of cocrystallized protein domains and unbound components of protein-protein complexes. Furthermore, ESCHER has been applied to the reassociation of secondary and supersecondary structure elements. The possibility of applying a docking method to the problem of protein structure prediction is discussed.

Protein Structure, Secondary↗

PubMed1996

HMG1 interacts with HOX proteins and enhances their DNA binding and transcriptional activation.

High mobility group protein 1 (HMG1) is a non-histone, chromatin-associated nuclear protein with a proposed role in the regulation of eukaryotic gene expression. We show that HMG1 interacts with proteins encoded by the HOX gene family by establishing protein-protein contacts between the HMG box domains and the HOX homeodomain. The functional role of these interactions was studied using the transcriptional activity of the human HOXD9 protein as a model. HMG1 enhances, in a dose-dependent fashion, the sequence-specific DNA binding activity in vitro, and the transcriptional activation in a co-transfection assay in vivo, of the HOXD9 protein. Functional interaction between HMG1 and HOXD9 is dependent on the DNA binding activity of the homeodomain, and requires the HOXD9 transcriptional activation domain. HMG1 enhances activation by HOXD9, but not by HOXD8, of the HOXD9-controlled element. Specific target recognition and functional interaction with HMG1 can be transferred to HOXD8 by homeodomain swapping. We propose that HMG1-like proteins might be general co-factors in HOX-mediated transcriptional activation, which facilitate access of HOX proteins to specific DNA targets, and/or introduce architectural constraints in the assembly of HOX-containing transcriptional complexes.

3T3 Cells↗

PubMed1995

Modelling antibody-antigen interactions: ferritin as a case study.

In this work, we propose a model for the structure of the antigen-antibody complex formed by human H-ferritin and an antibody that specifically recognizes it. We cloned and sequenced the antibody gene, predicted the antibody three-dimensional structure, and reconstructed the H-ferritin-antibody complex using an automated docking procedure previously validated on known complexes. This procedure allowed us to identify one putative complex which we carefully analysed, in order both to evaluate its likelihood, in light of a set of experimental results described in the literature, and to predict precisely which are the sites of interaction between the two molecules. Our model is compatible with the experimentally determined characteristics of the complex. Some of the residues that form the predicted antigenic site of ferritin can be found in the amino acid sequence of peptides selected from a random peptide library because of their affinity for the ferritin monoclonal antibody. Furthermore, the structural difference between the antigenic site in human H-ferritin and the corresponding region in other species permits us to rationalize the inability of the antibody to recognize human L-ferritin and rat, chicken and mouse H-ferritin. Through the analysis of our model complex, we identify a number of other residues putatively involved in the interaction. This multidisciplinary approach shows that synergy between computational and experimental methods may bring further insight into the understanding of antibody-antigen recognition rules.

Amino Acid Sequence↗

PubMed1994

PUZZLE: a new method for automated protein docking based on surface shape complementarity.

We describe here a novel procedure for automated protein docking, based only on geometric criteria. In our algorithm we project protein surfaces into bi-dimensional matrices; the search for complementary regions is performed by detecting matching sub-matrices. An exhaustive sampling of the rotation space is made in order to analyse all the possible relative orientations of the two proteins, but nevertheless this procedure requires a relatively short processing time (3 h to 24 h cpu time on a SG4D320, depending on the complexity of the input information). When tested with co-crystallized, free components and models of components of known protein-protein complexes, the method gave very satisfactory results. The procedure selects no more than four relative orientations of the molecular components, but the correct orientation is always present among them, ranking either first or second. In more than half the cases the "wrong" solutions nevertheless correctly identify most of the residues involved in the interaction. This is remarkable also in view of the fact that the chosen test complexes (trypsin-trypsin inhibitor and antibody-lysozyme) have a very different geometry of surface complementarity: trypsin inhibitor inserts a long side-chain into the deep specificity pocket of the protease, while the interface between antibody and lysozyme is rather flat and contains buried water molecules (not included in the calculation). In order to simulate a more realistic protein docking problem, we also used a trypsin inhibitor and an anti-lysozyme antibody model in our simulations, again with satisfying results.

Algorithms↗

PubMed1994

Molecular models and structural comparisons of native and mutant class I filamentous bacteriophages Ff (fd, f1, M13), If1 and IKe.

The filamentous bacteriophages are flexible rods about 1 to 2 microns long and 6 nm in diameter, with a helical shell of protein subunits surrounding a DNA core. The approximately 50-residue coat protein subunit is largely alpha-helix and the axis of the alpha-helix makes a small angle with the axis of the virion. The protein shell can be considered in three sections: the outer surface, occupied by the N-terminal region of the subunit, rich in acidic residues that interact with the surrounding solvent and give the virion a low isoelectric point; the interior of the shell, including a 19-residue stretch of apolar side-chains, where protein subunits interact mainly with each other; and the inner surface, occupied by the C-terminal region of the subunit, rich in basic residues that interact with the DNA core. The fact that virtually all protein side-chain interactions are between different subunits in the coat protein array, rather than within subunits, makes this a useful model system for studies of interactions between alpha-helix subunits in a macromolecular assembly. We describe molecular models of the class I filamentous bacteriophages. This class includes strains fd, f1, M13 (these 3 very similar strains are members of the Ff group), If1 and IKe. Our model of fd has been refined to fit quantitative X-ray fibre diffraction data to 30 A resolution in the meridional direction and 7 A resolution in the equatorial direction. A simulated 3.3 A resolution diffraction pattern from this model has the same general distribution of intensity as the experimental diffraction pattern. The observed diffraction data at 7 A resolution are fitted much better by the calculated diffraction pattern of our molecular model than by that of a model in which the alpha-helix subunit is represented by a rod of uniform density. The fact that our fd model explains the fd diffraction data is only part of our structure analysis. The atomic details of the model are supported by non-diffraction data, in part previously published and in part newly reported here. These data include information about permitted or forbidden side-chain replacements, about the effect of chemical modification, and about spectroscopic experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Control of ColE1 plasmid replication by antisense RNA.

One of the two major classes of regulatory strategies that control plasmid copy number involves recognition via base pairing between two plasmid-encoded complementary RNAs. The detailed analysis of this control circuitry has revealed some features of regulatory mechanisms based on RNA-RNA interaction that distinguish them from those based on protein-nucleic acid interaction. These features provide a framework with which to understand other regulatory mechanisms based on RNA-RNA interaction, and will aid in the design of efficient artificial antisense RNA systems.

Bacterial Proteins↗

PubMed1988

Control of ColE1 replication: low affinity specific binding of Rop (Rom) to RNAI and RNAII.

We have studied the interactions between the three molecules Rop, RNAI and RNAII that are involved in the regulatory mechanism controlling the replication of ColE1 plasmids. We show that it is possible to purify the two RNA molecules by passing an RNA mixture through an affinity column containing Rop immobilized to a solid support. The dissociation constants of the Rop-RNAI and Rop-RNAII complexes are of the order of 10(-4) M, several orders of magnitude higher than dissociation constants of stable protein-nucleic acid complexes (10(-10) M in the lambda repressor system). Although complete RNAI molecules have higher affinity, stem-and-loop I alone can also bind Rop, suggesting that this structure plays an important role in the interaction. Rop protects the stems of RNAI and RNAII from digestion by RNases while the sensitivity of the loops to digestion by RNase T1 is not affected by high concentrations of Rop. We propose a model for Rop-RNAI/RNAII interaction in which the dimeric protein acts as an adaptor between stem structures to position the two RNAs in the correct position for loop interaction.

Bacteriocin Plasmids↗

PubMed1983

Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria.

The sequence of the apocytochrome b (cob) gene of Neurospora crassa has been determined. The structural gene is interrupted by two intervening sequences of approximately 1260 bp each. The polypeptide encoded by the exons shows extensive homology with the cob proteins of Aspergillus nidulans and Saccharomyces cerevisiae (79% and 60%, respectively). The two introns are, however, located at sites different from those of introns in the cob genes of A. nidulans and S. cerevisiae (which contain highly homologous introns at the same site within the gene). The introns share several short regions of sequence homology (10-12 bp long) with each other and with other fungal mitochondrial introns. Moreover, the second intron contains a 50 nucleotide long sequence that is highly homologous with sequences within every ribosomal intron of fungal mitochondria sequenced to date. The conserved sequences may allow the formation of a core secondary structure, which is nearly identical in many mitochondrial introns. The conserved secondary structure may be required for intron splicing. The second intron contains an open reading frame, continuous with the preceding exon, of approximately 290 codons. Two stretches of 10 amino acid residues, conserved in many introns, are present in the open reading frame.

Amino Acid Sequence↗