Contribution of leucine 85 to the structure and function of Saccharomyces cerevisiae iso-1 cytochrome c.

Cytochrome c is a small electron-transport protein whose major role is to transfer electrons between complex III (cytochrome reductase) and complex IV (cytochrome c oxidase) in the inner mitochondrial membrane of eukaryotes. Cytochrome c is used as a model for the examination of protein folding and structure and for the study of biological electron-transport processes. Amongst 96 cytochrome c sequences, residue 85 is generally conserved as either isoleucine or leucine. Spatially, the side chain is associated closely with that of the invariant residue Phe82, and this interaction may be important for optimal cytochrome c activity. The functional role of residue 85 has been examined using six site-directed mutants of Saccharomyces cerevisiae iso-1 cytochrome c, including, for the first time, kinetic data for electron transfer with the principle physiological partners. Results indicate two likely roles for the residue: first, heme crevice resistance to ligand exchange, sensitive to both the hydrophobicity and volume of the side chain; second, modulation of electron-transport activity through maintenance of the hydrophobic character of the protein in the vicinity of Phe82 and the exposed heme edge, and possibly of the ability of this region to facilitate redox-linked conformational change.

(E)-1-(2'-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-(2-iodovinyl)benzene.

This analysis of the title compound, C13H13F2IO3, establishes the orientation of (E)-5-(CH=CH-I) as antiperiplanar (ap) to the C-C bond (5-6 position) of the 2,4-difluorophenyl ring system, with the (E)-5-(CH=CH-I) H atom located in close proximity (2.17 A) to the F4 atom of the 2,4-difluorophenyl moiety.

A tale of two charges: distinct roles for an acidic and a basic amino acid in the structure and function of cytochrome c.

Cytochrome c is a small electron transport protein found in the intermembrane space of mitochondria. As it interacts with a number of different physiological partners in a specific fashion, its structure varies little over eukaryotic evolutionary history. Two highly conserved residues found within its sequence are those at positions 13 and 90 (numbering is based on the standard horse cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and residue 90 is either Glu or Asp. There have been conflicting views on the roles to be ascribed to these residues, particularly residue 13, so the functional properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1 cytochrome c have been examined. Results indicate that the two residues do not interact specifically with each other; however, residue 13 (Arg) is likely to be involved in interactions between cytochrome c and other electrostatically oriented physiological partners (intermolecular), whereas residue 90 (Asp) is involved in maintaining the intrinsic structure and stability of cytochrome c (intramolecular). This is supported by molecular dynamics simulations carried out for these mutants where removal of the negative charge at position 90 leads to significant shifts in the conformations of neighboring residues, particularly lysine 86. Both charged residues appear to exert their effects through electrostatics; however, biological activity is significantly more sensitive to substitutions of residue 13 than of residue 90.

Electrostatic reversal of serine proteinase substrate specificity.

Mouse granzyme B is a member of the chymotrypsin family of serine proteinases that has an unusual preference for cleavage of substrates following aspartate residues. We show here that granzyme B can be redesigned by a single amino acid substitution in one wall of the specificity pocket, arginine-226 to glutamate, to hydrolyze preferentially thioester substrates following basic amino acids. Amide substrates, however, were not hydrolyzed by the variant granzyme B. These results show that residue 226 is a primary determinant of granzyme B specificity and imply that additional structural components are required for catalysis of amide bonds. Molecular modeling indicated subtle variation in glutamate-226 orientation depending upon the state of protonation of the gamma-carboxylate, which may account for the secondary specificity of this enzyme for substrates containing phenylalanine. This represents the first example of electrostatic reversal of serine proteinase substrate specificity and suggests that residue 226 is a primary substrate specificity determinant in the granzyme B lineage of serine proteinases.

Lowering the entropic barrier for binding conformationally flexible inhibitors to enzymes.

The design of inhibitors with enhanced potency against proteolytic enzymes has many applications for the treatment of human diseases. In addition to the optimization of chemical interactions between the enzyme and inhibitor, the binding affinity can be increased by constraining the inhibitor to the conformation that is recognized by the enzyme, thus lowering the entropic barrier to complex formation. We have structurally characterized the complexes of a macrocyclic pentapeptide inhibitor and its acyclic analogue with penicillopepsin, an aspartic proteinase, to study the effect of conformational constraint on the binding affinity. The phosphonate-based macrocycle PPi4 (Ki = 0.10 nM) is covalently linked at the P2-Asn and P1'-Phe side chains [nomenclature of Schechter and Berger, Biochim. Biophys. Res. Commun. (1967) 27, 157-162] via an amide bond, relative to the acyclic compound PPi3 (Ki = 42 nM). Comparisons of the high-resolution crystal structures of PPi4-penicillopepsin (0.95 A) and PPi3-penicillopepsin (1.45 A) reveal that the conformations of the inhibitors and their interactions with the enzyme are similar. The 420-fold increase in the binding affinity of PPi4 is attributed to a reduction in its conformational flexibility, thus providing the first rigorous measure of the entropic contribution to the binding energy in a protein-ligand complex and stressing the advantages of the design strategy.

Synergy in protein engineering. Mutagenic manipulation of protein structure to simplify semisynthesis.

Semisynthesis is a chemical technique of protein engineering that provides a valuable complement to directed mutagenesis. It is the method of choice when the structural modification requires, for example, a noncoded amino acid. The process involves specific and limited protein fragmentation, structural manipulation of the target sequence, and subsequent religation of fragments to give the mutant holoprotein. We suggested and demonstrated that mutagenesis and semisynthesis could be used synergistically to achieve protein engineering goals otherwise unobtainable, if mutagenesis was used to shuffle methionine residues in the yeast cytochrome c sequence (Wallace, C. J. A., Guillemette, J. G., Hibiya, Y., and Smith, M. (1991) J. Biol. Chem. 266, 21355-21357). These residues can not only be sites of specific cleavage by CNBr but also of spontaneous peptide bond synthesis between fragments in noncovalent complexes, which greatly facilitates the semisynthetic process. We have now used an informed "methionine scan" of the protein sequence to discover other useful sites and to characterize the factors that promote this extraordinary and convenient autocatalytic religation. Of eight sites canvassed, in a wide range of settings, five efficiently provoked peptide bond synthesis. The principal factor determining efficiency seems to be the hydropathy of the religation site. The mutants created have also provided some new insights on structure-function relationships in the cytochrome.

Definition of a nucleotide binding site on cytochrome c by photoaffinity labeling.

We have used TNP-8N3-AMP (2'(3')-O-(2,4,6-trinitrophenyl)-8-azidoadenosine monophosphate) and TNP-8N3-ATP to probe the ATP binding site(s) of cytochrome c. Irradiation of cytochrome c with close to stoichiometric amounts of TNP-8N3-AMP at low ionic strength derivatized approximately half of the protein, with the mono-derivatized species being associated with four peaks (B, 6%; C, 17%; D, 24%; E, 4%) eluted from a cation exchange column. Irradiation in the presence of ATP suggested that the main peaks C and D resulted from more specific nucleotide binding. Thermolysin digestion and TNP-peptide purification and sequencing revealed that peak C was associated with derivatization of mainly Lys-86 and to a lesser extent Lys-72 and peak D with mainly Lys-87 and less so with Lys-72. Minor peaks B and E could not be identified. TNP-8N3-ATP photolabeling produced similar results, showing favored interaction of the adenyl ring with Lys-86 and Lys-87 and to a lesser extent with Lys-72. The results are compatible with previous findings that suggest that the principal locus of ATP binding is at nearby Arg-91 (Corthesy, B. E., and Wallace, C. J. A.(1986) Biochem. J. 236, 359-364). Molecular modeling with energy-minimized docking of ATP between the 60s helix and the 80s stretch with the gamma-phosphate constrained to interact with Arg-91, places the 8 position close to Lys-86 and Lys-87 in the anti conformation about the glycosidic bond and to Lys-72 in the syn conformation, and the ribose hydroxyls within H-bonding distance of Glu-69.