Conformational investigation of alpha,beta-dehydropeptides. XVI. Beta-turn tendency in Ac-Pro-DeltaXaa-NHMe: crystallographic and theoretical studies.

The crystal structures of two diastereomeric alpha,beta-dehydrobutyrine peptides Ac-Pro-(Z)-DeltaAbu-NHMe (I) and Ac-Pro-(E)-DeltaAbu-NHMe (II) have been determined. Both dehydropeptides adopt betaI-turn conformation characterized by the pairs of (phi(i+1), psi(i+1)) and (phi(i+2), psi(i+2)) angles as -66, -19, -97, 11 degrees for I and -59, -27, -119, 29 degrees for II. In each peptide, the betaI turn is stabilized by (i + 3) --> i intramolecular hydrogen bonds with N...O distance of 3.12 A for I and 2.93 A for II. These structures have been compared to the crystal structures of homologous peptides Ac-Pro-DeltaVal-NHMe and Ac-Pro-DeltaAla-NHMe. Theoretical analyses by DFT/B3LYP/6-31 + G** method of conformers formed by these four peptides and by the saturated peptide Ac-Pro-Ala-NHMe revealed that peptides with a (Z) substituent at the C(beta) (i+2) atom of dehydroamino acid, i.e. Ac-Pro-DeltaVal-NHMe and Ac-Pro-(Z)-DeltaAbu-NHMe, predominantly form beta turns, both in vacuo and in polar environment. The tendency to adopt beta-turn conformation is much weaker for the peptides lacking the (Z) substituent, Ac-Pro-(E)-DeltaAbu-NHMe and Ac-Pro-DeltaAla-NHMe. The latter adopts a semi-extended or an extended conformation in every polar environment, including a weakly polar solvent. The saturated peptide Ac-Pro-Ala-NHMe in vacuo prefers a beta-turn conformation, but in polar environment the differences between various conformers are small. The role of pi-electron correlation and intramolecular hydrogen bonds interaction in stabilizing the hairpin structures are discussed.

How dihydrolipoamide dehydrogenase-binding protein binds dihydrolipoamide dehydrogenase in the human pyruvate dehydrogenase complex.

The dihydrolipoamide dehydrogenase-binding protein (E3BP) and the dihydrolipoamide acetyltransferase (E2) component enzyme form the structural core of the human pyruvate dehydrogenase complex by providing the binding sites for two other component proteins, dihydrolipoamide dehydrogenase (E3) and pyruvate dehydrogenase (E1), as well as pyruvate dehydrogenase kinases and phosphatases. Despite a high similarity between the primary structures of E3BP and E2, the E3-binding domain of human E3BP is highly specific to human E3, whereas the E1-binding domain of human E2 is highly specific to human E1. In this study, we characterized binding of human E3 to the E3-binding domain of E3BP by x-ray crystallography at 2.6-angstroms resolution, and we used this structural information to interpret the specificity for selective binding. Two subunits of E3 form a single recognition site for the E3-binding domain of E3BP through their hydrophobic interface. The hydrophobic residues Pro133, Pro154, and Ile157 in the E3-binding domain of E3BP insert themselves into the surface of both E3 polypeptide chains. Numerous ionic and hydrogen bonds between the residues of three interacting polypeptide chains adjacent to the central hydrophobic patch add to the stability of the subcomplex. The specificity of pairing for human E3BP with E3 is interpreted from its subcomplex structure to be most likely due to conformational rigidity of the binding fragment of the E3-binding domain of E3BP and its exquisite amino acid match with the E3 target interface.

Structural basis for the catalytic activity of human serine/threonine protein phosphatase-5.

Serine/threonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth and cellular responses to stress. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 A. From this structure we propose a mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a conserved Asp271-M1:M2-W1-His427-His304-Asp274 catalytic motif (where M1 and M2 are metals and W1 is a water molecule). The structure of PP5c provides a structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

Function of several critical amino acids in human pyruvate dehydrogenase revealed by its structure.

Pyruvate dehydrogenase (E1), an alpha(2)beta(2) tetramer, catalyzes the oxidative decarboxylation of pyruvate and reductive acetylation of lipoyl moieties of the dihydrolipoamide acetyltransferase. The roles of betaW135, alphaP188, alphaM181, alphaH15, and alphaR349 of E1 determined by kinetic analysis were reassessed by analyzing the three-dimensional structure of human E1. The residues identified above are found to play a structural role rather than being directly involved in catalysis: betaW135 is in the center of the hydrophobic interaction between beta and beta' subunits; alphaP188 and alphaM181 are critical for the conformation of the TPP-binding motif and interaction between alpha and beta subunits; alphaH15 is necessary for the organization of the N-terminus of alpha and alpha' subunits; and alphaR349 supports the interaction of the C-terminus of the alpha subunits with the beta subunits. Analysis of several critical E1 residues confirms the importance of residues distant from the active site for subunit interactions and enzyme function.

Structural basis for flip-flop action of thiamin pyrophosphate-dependent enzymes revealed by human pyruvate dehydrogenase.

The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the Calpha-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-A resolution. We propose a model for the flip-flop action of this enzyme through a concerted approximately 2-A shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.