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Metallocarboxypeptidases and their protein inhibitors: Structure, function and biomedical properties

Among the different aspects of recent progress in the field of metallocarboxypeptidases has been the elucidation of the three dimensional structures of the pro-segments (in monomeric or oligomeric species) and their role in the expression, folding and inhibition/activation of the pancreatic and pancreatic-like forms. Also of great significance has been the cloning and characterization of several new regulatory carboxypeptidases, enzymes that are related with important functions in protein and peptide processing and that show significant structural differences among them and also with the digestive ones. Many regulatory carboxypeptidases lack a pro-region, unlike the digestive forms or others in between from the evolutionary point of view. Finally, important advances have been made on the finding and characterization of new protein inhibitors of metallocarboxypeptidases, some of them with interesting potential applications in the biotechnological/biomedical fields. These advances are analyzed here and compared with the earlier observations in this field, which was first explored by Hans Neurath and collaborators.

Mycoplasma genitalium P140 and P110 Cytadhesins Are Reciprocally Stabilized and Required for Cell Adhesion and Terminal-Organelle Development

Mycoplasma genitalium is a human pathogen that mediates cell adhesion by a complex structure known as the attachment organelle. This structure is composed of cytadhesins and cytadherence-associated proteins, but few data are available about the specific role of these proteins in M. genitalium cytadherence. We have deleted by homologous recombination the mg191 and mg192 genes from the MgPa operon encoding the P140 and P110 cytadhesins. Molecular characterization of these mutants has revealed a reciprocal posttranslational stabilization between the two proteins. Loss of either P140 or P110 yields a hemadsorption-negative phenotype and correlates with decreased or increased levels of cytoskeleton-related proteins MG386 and DnaK, respectively. Scanning electron microscopy analysis reveals the absolute requirement of P140 and P110 for the proper development of the attachment organelle. The phenotype described for these mutants resembles that of the spontaneous class I and class II cytadherence-negative mutants [G. R. Mernaugh, S. F. Dallo, S. C. Holt, and J. B. Baseman, Clin. Infect. Dis. 17(Suppl. 1):S69-S78, 1993], whose genetic basis remained undetermined until now. Complementation assays and sequencing analysis demonstrate that class I and class II mutants are the consequence of large deletions affecting the mg192 and mg191-mg192 genes, respectively. These deletions originated from single-recombination events involving sequences of the MgPa operon and the MgPa island located immediately downstream. We also demonstrate the translocation of MgPa sequences to a particular MgPa island by double-crossover events. Based on these observations, we propose that in addition to being a source of antigenic variation, MgPa islands could be also involved in a general phase variation mechanism switching on and off, in a reversible or irreversible way, the adhesion properties of M. genitalium.

Crystal structure of Bacillus licheniformis 1,3-1,4-β-d-glucan 4-glucanohydrolase at 1.8 Å resolution

The crystal structure of the 1,3‐1,4‐β‐d‐glucan 4‐glucanohydrolase from Bacillus licheniformis is solved at a resolution of 1.8 Å and refined to R = 16.5%. The protein has a similar β‐sandwich structure as the homologous enzyme from Bacillus macerans and the hybrid H(A16‐M). This demonstrates that the jellyroll fold of these proteins is remarkably rigid and only weakly influenced by crystal contacts. The crystal structure permits to extend mechanistic considerations derived for the B. licheniformis enzyme to the entire class of bacterial 1,3‐1,4‐β‐d‐glucan 4‐glucanohydrolases.

ArchDB: automated protein loop classification as a tool for structural genomics

The annotation of protein function has become a crucial problem with the advent of sequence and structural genomics initiatives. A large body of evidence suggests that protein structural information is frequently encoded in local sequences, and that folds are mainly made up of a number of simple local units of super-secondary structural motifs, consisting of a few secondary structures and their connecting loops. Moreover, protein loops play an important role in protein function. Here we present ArchDB, a classification database of structural motifs, consisting of one loop plus its bracing secondary structures. ArchDB currently contains 12,665 super-secondary elements classified into 1496 motif subclasses. The database provides an easy way to retrieve functional information from protein structures sharing a common motif, to search motifs found in a given SCOP family, superfamily or fold, or to search by keywords on proteins with classified loops. The ArchDB database of loops is located at http://sbi.imim.es/archdb.

Glycoprotein E of bovine herpesvirus type 1 is involved in virus transmission by direct cell-to-cell spread

In order to identify the role of the bovine herpesvirus type 1 (BHV-1) glycoprotein E (gE) in the viral infection cycle, we have constructed a BHV-1 gE deletion mutant strain (BHV-1 gE-). This strain was assayed in vitro by comparing its growth kinetics with the wild type strain used as a host of the deletion. Our results indicate that those conditions which prevent the infection by direct adsorption to the cells (presence of a semi-solid medium or presence of neutralizing antibodies in the medium) selectively inhibit the growth of the gE- strain, suggesting that gE plays a central role in the BHV-1 spread by direct cell-to-cell transmission, a major mechanism of the BHV-1 in vivo virulence.

Modelling repressor proteins docking to DNA

The docking of repressor proteins to DNA starting from the unbound protein and model‐built DNA coordinates is modeled computationally. The approach was evaluated on eight repressor/DNA complexes that employed different modes for protein/ DNA recognition. The global search is based on a protein‐protein docking algorithm that evaluates shape and electrostatic complementarity, which was modified to consider the importance of electrostatic features in DNA‐protein recognition. Complexes were then ranked by an empirical score for the observed amino acid /nucleotide pairings (i.e., protein‐DNA pair potentials) derived from a database of 20 protein/DNA complexes. A good prediction had at least 65% of the correct contacts modeled. This approach was able to identify a good solution at rank four or better for three out of the eight complexes. Predicted complexes were filtered by a distance constraint based on experimental data defining the DNA footprint. This improved coverage to four out of eight complexes having a good model at rank four or better. The additional use of amino acid mutagenesis and phylogenetic data defining residues on the repressor resulted in between 2 and 27 models that would have to be examined to find a good solution for seven of the eight test systems. This study shows that starting with unbound coordinates one can predict three‐dimensional models for protein/DNA complexes that do not involve gross conformational changes on association. Proteins 33:535–549, 1998.

Cell division in a minimal bacterium in the absence of ftsZ

Mycoplasma genomes exhibit an impressively low amount of genes involved in cell division and some species even lack the ftsZ gene, which is found widespread in the microbial world and is considered essential for cell division by binary fission. We constructed a Mycoplasma genitalium ftsZ null mutant by gene replacement to investigate the role of this gene and the presence of alternative cell division mechanisms in this minimal bacterium. Our results demonstrate that ftsZ is non‐essential for cell growth and reveal that, in the absence of the FtsZ protein, M. genitalium can manage feasible cell divisions and cytokinesis using the force generated by its motile machinery. This is an alternative mechanism, completely independent of the FtsZ protein, to perform cell division by binary fission in a microorganism. We also propose that the mycoplasma cytoskeleton, a complex network of proteins involved in many aspects of the biology of these microorganisms, may have taken over the function of many genes involved in cell division, allowing their loss in the regressive evolution of the streamlined mycoplasma genomes.

Properties of a novel glucose-enhanced β-glucosidase purified from Streptomyces sp. (ATCC 11238)

An inducible intracellular beta-glucosidase (EC 3.2.1.21) from Streptomyces sp. QM-B814 (ATCC 11238) has been purified and characterized. The purified polypeptide is monomeric with a relative molecular mass of 62 kDa by SDS-PAGE and 42 kDa by size-exclusion chromatography; its isoelectric point is 4.2. The difference in the molecular mass values can be attributed to the glycosylated nature of the protein. The purified enzyme has a pH optimum of 6.0-6.5. The temperature optimum for activity is 50 degrees C; at this temperature the enzyme is stable for 1 h. The enzyme hydrolyzes mainly aryl-beta-glucosides but also presents significant activity against beta-linked disaccharides and maltose. The enzyme displays an unusual kinetic behavior and biphasic Lineweaver-Burk and Eadie-Hofstee plots for p-nitrophenyl-beta-D-glucoside and cellobiose were obtained. The enzyme presents beta-glycosyltransferase activity and an exoglycosidase-type action on cellodextrins. It is inhibited by delta-gluconolactone (Ki 0.44 mM) but, remarkably, glucose in the range 25-200 mM enhances the rate of p-nitrophenyl-beta-D-glucoside hydrolysis.

MultitaskProtDB: a database of multitasking proteins

We have compiled MultitaskProtDB, available online at http://wallace.uab.es/multitask, to provide a repository where the many multitasking proteins found in the literature can be stored. Multitasking or moonlighting is the capability of some proteins to execute two or more biological functions. Usually, multitasking proteins are experimentally revealed by serendipity. This ability of proteins to perform multitasking functions helps us to understand one of the ways used by cells to perform many complex functions with a limited number of genes. Even so, the study of this phenomenon is complex because, among other things, there is no database of moonlighting proteins. The existence of such a tool facilitates the collection and dissemination of these important data. This work reports the database, MultitaskProtDB, which is designed as a friendly user web page containing >288 multitasking proteins with their NCBI and UniProt accession numbers, canonical and additional biological functions, monomeric/oligomeric states, PDB codes when available and bibliographic references. This database also serves to gain insight into some characteristics of multitasking proteins such as frequencies of the different pairs of functions, phylogenetic conservation and so forth.

Expression of a synthetic gene encoding potato carboxypeptidase inhibitor using a bacterial secretion vector

A synthetic gene encoding the 39-amino-acid (aa) potato carboxypeptidase inhibitor IIa (PCI-IIa) has been constructed and expressed using the secretion vector, pIN-III-ompA-3, fused in frame to the OmpA signal peptide-encoding sequence. Recombinant Escherichia coli secreted a PCI with 10 additional aa at the N terminus (rePCI + 10). These extra aa were removed by site-directed mutagenesis giving a PCI with no additional aa (rePCI), as shown by fast atom bombardment mass spectrometry (M(r) 4295). The two forms of rePCI were found almost exclusively in the culture medium, not in the periplasmic space, as would be expected from OmpA signal peptide fusions. Both rePCI + 10 and rePCI are biologically active and react strongly with serum raised against PCI from potato. A method for the purification of rePCI to homogeneity has been developed. The purified rePCI shows a Ki for carboxypeptidase A within the range of the natural PCI-IIa (1.5-2.7 nM). These results indicate that both rePCI + 10 and rePCI are properly folded and that their three disulfide bridges are correctly formed. Together with previous reports, our results show that fusion to a secretion signal peptide is an effective way of producing small proteins containing disulfide bridges in a biologically active form.