A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity

A three-dimensional structure for human cytochrome P450IA1 waspredicted based on the crystal coordinates of cytochrome P450camfrom Pseudomonas putida. As there was only 15% residue identitybetween the two enzymes, additional information was used toestablish an accurate sequence alignment that is a prerequisitefor model building. Twelve representative eukaryotic sequenceswere aligned and a net prediction of secondary structure wasmatched against the known -helices and ß-sheets ofP450cam. The cam secondary structure provided a fixed main-chainframework onto which loops of appropriate length from the humanP450IA1 structure were added. The model-built structure of thehuman cytochrome conformed to the requirements for the segregationof polar and nonpolar residues between the core and the surface.The first 44 residues of human cytochrome P450 could not bebuilt into the model and sequence analysis suggested that residues1–26 formed a single membrane-spanning segment. Examinationof the sequences of cytochrome P450s from distinct gene familiessuggested specific residues that could account for the differencesin substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene,was fitted into the proposed active site and this planar aromaticmolecule could be accommodated into the available cavity. Residuesthat are likely to interact with the haem were identified. Thesequence similarity between 59 eukaryotic enzymes was representedas a dendrogram that in general clustered according to genefamily. Until a crystallographic structure is available, thismodel-building study identifies potential residues in cytochromeP450s important in the function of these enzymes and these residuesare candidates for site-directed mutagenesis.     

A detailed molecular model for human aromatase

Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P-450arom (human aromatase) based on that of P-450cam, the only cytochrome P-450 enzyme for which the crystal structure is known. The predicted structure is found to be in good agreement with current experimental data; both direct, from site-directed mutagenesis studies, and indirect, from the consideration of the structures and activities of known substrates and inhibitors.     

Prediction and analysis of SH2 domain-phosphopeptide interactions

Src homology 2 (SH2) domains are small protein modules of -100amino acids that are found in many proteins involved in intracellularsignal transduction. They mediate protein-protein interactionsand modulate enzyme activity by their ability to bind to specificsequence patterns that contain a phosphorylated tyrosine. Asthe three-dimensional structures of the phosphatidylinositol(PI) 3-kinase, Lck, Src and Abl SH2 domains have been shownto be similar, we have modelled other SH2 domains that showdistinct sequence specificity to allow comparative analysisof SH2-phosphopeptide interactions. The SH2 domains of PLC-Nterm.,Nck, Grb2, GAP and Abl have been model-built with high-affinityphosphopeptides fitted into the putative binding sites. Foreach SH2 domain a detailed analysis of the peptide-protein interactionwas performed. It is apparent that specificity is mainly conferredby three to five residues downstream from the phosphotyrosineresidue (Y*), especially, although not exclusively, peptideposition Y* + 3. The SH2 pocket that binds the Y* + 3 residueis mainly composed of three sections: part of strand (ßEgoing into loop EF, part of B and loop BG. The residues thatconstitute the Y* +3 binding pocket show variability that seemsto determine which amino acid binds preferentially. Residueposition ßE4 seems to play a vital role in the SH2specificity. This study shows that the development of modellingprotocols for SH2 domains whose structure has not been determinedcan prove very useful in predicting which residues are involvedin conferring the affinity and binding specificity of thesedomains towards distinct phosphotyrosine-containing sequences.     

Analysis and prediction of the location of catalytic residues in enzymes

The catalytic residues of an enzyme are defined as the aminoacids directly involved in chemical catalysis. They mainly actas a general acid–base, electrophilic or nucleophiliccatalyst or they polarize and stabilize the transition state.An analysis of the structural features of 36 catalytic residuesin 17 enzymes of known structure and with defined mechanismis reported. Residues that bind metal ions (Zn² and Cu²) areconsidered separately. The features examined are: residue type,location in secondary structure, separation between the residues,accessibility to solvent, intra-protein electrostatic interactions,mobility as evaluated from crystallographic temperature factors,polarity of the environment and the sequence conservation betweenhomologous enzymes of residues that were sequentially or spatiallyclose to the catalytic residue. In general the environment ofcatalytic residues is similar to that of polar side chains thathave low accessibility to solvent. Two algorithms have beendeveloped to identify probable catalytic residues. Scanningan alignment of homologous enzyme sequences for peaks of sequenceconservation identifies 13 out of the 16 catalytic residueswith 50 residues overpredicted. When the conservation of thespatially close residues is used instead, a different set of13 residues are identified with 47 residues overpredicted. Acombination of the two algorithms identifies 11 residues with36 residues overpredicted.     

1H NMR structure of an antifungal gamma-thionin protein SIalpha1: similarity to scorpion toxins

The three-dimensional structure of the Sorghum bicolor seed protein gamma-thionin SIalpha1 has been determined by 2D 1H nuclear magnetic resonance (NMR) spectroscopy. The secondary structure of this 47-residue antifungal protein with four disulphide bridges consists of a three-stranded antiparallel sheet and one helix. The helix is tethered to the sheet by two disulphide bridges which link two successive turns of the helix to alternate residues i, i+2 in one strand. Possible binding sites for antifungal activity are discussed. The same fold has been observed previously in several scorpion toxins.