Interactions of Streptomyces griseus aminopeptidase with a methionine product analogue: a structural study at 1.53 A resolution.

SGAP is an aminopeptidase present in the extracellular fluid of Streptomyces griseus cultures. It is a double-zinc enzyme with a strong preference for large hydrophobic amino-terminus residues. It is a monomeric (30 kDa) heat-stable enzyme, with a high and efficient catalytic activity modulated by calcium ions. The small size, high activity and heat stability make SGAP a very attractive enzyme for various biotechnological applications. Only one other related aminopeptidase (Aeromonas proteolytica AP; AAP) has been structurally analyzed to date and its structure was shown to be considerably similar to SGAP, despite the low sequence homology between the two enzymes. The motivation for the detailed structural analysis of SGAP originated from a strong mechanistic interest in the family of double-zinc aminopeptidases, combined with the high potential applicability of these enzymes. The 1.75 A crystallographic structure of native SGAP has been previously reported, but did not allow critical mechanistic interpretations owing to inconclusive structural regions around the active site. A more accurate structure of SGAP at 1.58 A resolution is reported in this paper, along with the 1.53 A resolution structure of the SGAP complex with inhibitory methionine, which is also a product of the SGAP catalytic process. These two high-resolution structures enable a better understanding of the SGAP binding mode of both substrates and products. These studies allowed the tracing of the previously disordered region of the enzyme (Glu196-Arg202) and the identification of some of the functional groups of the enzyme that are involved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and Phe219). These studies also suggest that Glu131 is directly involved in the catalytic mechanism of SGAP, probably as the hydrolytic nucleophile. The structural results are compared with a recent structure of AAP with an hydroxamate inhibitor in order to draw general functional conclusions which are relevant for this family of low molecular-weight aminopeptidases.

Importance of anchor group positioning in protein loop prediction.

The aim of loop prediction in protein homology modeling is to connect the main chain ends of two successive regions, conserved in template and target structures by protein fragments that are as similar to the target as possible. For the development of a new loop prediction method, examples of insertions and deletions were searched automatically in data sets of structurally aligned protein pairs. Three different criteria were applied for the determination of the positions where the main chain conformations of the proteins begin to differ, i.e., the anchoring groups of the insertions and deletions, giving three test data sets. The target structures in these data sets were predicted by inserting fragments from different fragment data banks between the anchoring groups of the templates. The proposals of matching fragments were sorted with decreasing correspondence in the geometry of the anchoring groups. For assessment of the prediction quality, the template loops were substituted by the proposed ones, and their root mean square deviations to the target structures were determined. In addition, the best 20 fragments in the whole loop data bank used-those with the lowest deviations from the target structures after insertion into the templates-were determined and compared with the proposals. The analysis of the results shows limitations of knowledge-based loop prediction. It is demonstrated that the selection of the anchoring groups is the most important step in the whole procedure. Proteins 1999;37:56-64.

Inhibition of Streptomyces griseus aminopeptidase and effects of calcium ions on catalysis and binding--comparisons with the homologous enzyme Aeromonas proteolytica aminopeptidase.

Streptomyces griseus aminopeptidase is a zinc metalloenzyme containing 2 mol zinc/mol protein, similar to the homologous enzyme Aeromonas proteolytica aminopeptidase. In addition, a unique Ca2+-binding site has been identified in the Streptomyces enzyme, which is absent in the Aeromonas enzyme. Binding of Ca2+ enhances stability of the Streptomyces enzyme and modulates its activity and affinity towards substrates and inhibitors in a structure-dependent manner. Among the three hydrophobic 4-nitroanilides of alanine, valine and leucine, the latter displays the largest overall activation (increase in k(cat)/Km). Large enhancements in affinity (1/Ki) upon Ca2+ binding have been observed for inhibitors with flexible (leucine-like) residues at their N-termini and smaller enhancements for inhibitors with rigid (phenylalanine-like) residues.

Creation and characterization of a new, non-redundant fragment data bank.

The success achieved for protein structure prediction of loop regions with insertions and deletions by knowledge-based methods depends on the quality of the underlying information, i.e. a fragment data bank as complete as possible is needed. However, the greater the number of proteins contributing to the data base the more redundant information is included, which leads to structurally similar proposals in loop predictions and to longer times for extracting fragments. So it is not only necessary to increase the number of proteins for building the loop data base but also to cluster the resulting fragments according to their structural similarities in order to remove redundancy. Here, a new, non-redundant fragment data bank is described, which is based on all proteins in the Brookhaven Protein Data Bank (release 7/95) with a resolution > or = 2.0 A and which can be updated easily by including new information from structures to be solved in the future. In the clustering process presented, the resulting clusters are optimized in several cycles until self-consistency. In this way all redundant information is removed without loosing any significantly different fragments. Finally the resulting fragment data bank is analysed with respect to its completeness.

NMR spectroscopic evidence that helodermin, unlike other members of the secretin/VIP family of peptides, is substantially structured in water.

The structure in water and additionally in 50% trifluoroethanol (TFE) solution of helodermin, an amidated peptide consisting of 35 amino acids, was elucidated by 2D 1H NMR spectroscopy initially from H alpha chemical shifts and qualitative NOE data. Detailed structures were calculated from the quantitative NOE data which were used as distance restraints in molecular dynamics and energy minimization calculations. Regions of stable secondary structure were defined from the resulting final peptide conformations using a new fitting program that takes into account the summed RMS differences between all structures for short segments of 2-5 residues in length. This procedure allows a reasonably objective method of defining the edges of stable structure. In contrast to other members of the secretin/VIP family of peptides, helodermin shows a defined secondary structure in water alone and possesses an alpha-helix from Glu-9 to Leu-23 that was further stabilized and slightly extended (Phe-6 to Ala-24) on addition of TFE. The N- and C-termini were unstructured in both solutions. Such features, in particular the observation of a linear helix 18 +/- 2 residues in length, are common to other members of the family and become more pronounced in hydrophobic environments. The data provide further circumstantial evidence that an alpha-helix conformation is necessary for receptor binding. The prolonged physiological action of helodermin, compared to its C-terminal deletion analogues and VIP, is at least in part due to the unusual stable secondary structure.

Similarities between protein 3-D structures.

A simple, new, systematic method has been developed to evaluate the structural similarity of proteins. Firstly, similar protein fragments of a definable length are detected, combined in pairs and superimposed. The fits are improved until a maximum number of C alpha atoms show a distance below a given threshold value. The final number of matching C alpha atoms can be used to quantify the similarity of protein folds and to identify the best superposition of two proteins. using this procedure a systematic search for structural similarities of proteins in the Brookhaven Protein Data Bank was performed. The results of these investigations allow us to divide the proteins into 182 structural families and to estimate the relationships between single members of the classes found.