Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases.

Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed 'RING/HECT hybrid' enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin's cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.

Structure of melanoma inhibitory activity protein, a member of a recently identified family of secreted proteins.

Melanoma inhibitory activity (MIA) is a 12-kDa protein that is secreted from both chondrocytes and malignant melanoma cells. MIA has been reported to have effects on cell growth and adhesion, and it may play a role in melanoma metastasis and cartilage development. We report the 1.4-A crystal structure of human MIA, which consists of an Src homology 3 (SH3)-like domain with N- and C-terminal extensions of about 20 aa. each. The N- and C-terminal extensions add additional structural elements to the SH3 domain, forming a previously undescribed fold. MIA is a representative of a recently identified family of proteins and is the first structure of a secreted protein with an SH3 subdomain. The structure also suggests a likely protein interaction site and suggests that, unlike conventional SH3 domains, MIA does not recognize polyproline helices.

The structural basis for pseudoreversion of the H95N lesion by the secondary S96P mutation in triosephosphate isomerase.

The structural basis for the 3000-fold decrease in catalytic efficiency of the H95N mutant chicken triosephosphate isomerase and the 60-fold regain of catalytic efficiency in the double mutant, H95N.S96P, have been analyzed. The results from a combination of X-ray crystallography and Fourier transform infrared spectroscopy experiments indicate that the predominant defect in the H95N mutant isomerase appears to be its inability to bind the substrate in a coplanar, cis conformation. The structures of each mutant isomerase were determined from X-ray crystallography of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog with the isomerase, and each was solved to a resolution of 1.9 A. The PGH appeared to be in two different conformations in which the enediol-mimicking atoms, O2-N2-C1-O1, of the PGH were not coplanar. No density was observed that would correspond to the coplanar conformation. Two bands are observed for the dihydroxyacetone phosphate carbonyl in the H95N mutant FTIR spectrum, and these can be explained if the O1 of DHAP, like the O1 of PGH in the crystal structure, is in two different positions. Two ordered water molecules are located between O1 of PGH and N delta of N95. Comparison of the structure of the pseudorevertant, H95N.S96P with that for the H95N single mutant, shows that S96P mutation causes the double mutant to regain the ability to bind PGH predominantly in the coplanar, cis conformation. Electron density for a single ordered water molecule bridging the N95 amide side chain and the O2 of PGH is observed, but the density was weak, perhaps indicating that the water molecule is somewhat disordered. Whether or not a water molecule is hydrogen bonded to O2 of PGH may explain the two carbonyl stretching frequencies observed for the GAP carbonyl. Together, the crystal structures and the FTIR data allow a complete explanation of the catalytic properties of these two mutant isomerases.

The structural basis for pseudoreversion of the E165D lesion by the secondary S96P mutation in triosephosphate isomerase depends on the positions of active site water molecules.

The structural basis for the improvement in catalytic efficiency of the mutant E165D chicken triosephosphate isomerase by the secondary mutation, S96P, has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. All X-ray structures were of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog, with the isomerase, and each was solved to a resolution of 1.9 A. Comparison of the structure of the double mutant, E165D.S96P, with that of the single mutant, E165D, as well as with the wild-type isomerase shows only insignificant differences in the positions of the side chains in all of the mutants when compared with the wild-type isomerase, except that in both the E165D and E165D.S96P mutants, the aspartate side chain was approximately 0.7 A further away from the substrate analog than the glutamate side chain. Significant differences were observed in the crystal structure of the E165D.S96P double mutant in the positions of ordered water molecules bound at the active site. The loss of two water molecules located near the side chain at position 165 was observed in isomerases containing the S96P mutation. The resulting increase in hydrophobicity of the pocket probably causes an increase in the pKa of the catalytic base, D165, thereby improving its basicity. A new ordered water molecule was observed underneath the bound PGH in the E165D.S96P structure, which likely decreases the pKa's of the substrate protons, thereby increasing their acidity. An enzyme derived carbonyl stretch at 1746 cm-1 that is only observed in the IR spectrum of the E165D.S96P double mutant isomerase with bound substrates has been assigned to a stable ground state protonated D165-enediol(ate) intermediate complex. Thus, the gain in activity resulting from the S96P second site change probably results from a combination of improving the basicity of the enzyme, improving the acidity of the substrate protons, and stabilization of a reaction intermediate. All three of these effects seem to be caused by changes in bound water molecules.

Thrombin inhibition by cyclic peptides from thrombomodulin.

Peptides corresponding to the loop regions of the fourth, fifth, and sixth epidermal growth factor (EGF)-like domains of thrombomodulin (TM) have been synthesized and assayed for thrombin inhibition, as indicated by both inhibition of thrombin-mediated fibrinogen clotting and inhibition of the association of thrombin with TM that results in protein C activation. Peptides from the fifth EGF-like domain showed significant inhibition of fibrinogen clotting and protein C activation, whereas peptides from the fourth and sixth EGF-like domains were weak inhibitors in both assays. Two structural features were important for inhibitory potency of the peptides from the fifth EGF-like domain: cyclization by a disulfide bond and attachment of the "tail" amino acids C-terminal to the disulfide loop. Linear control peptides did not significantly inhibit clotting or protein C activation. The C-terminal loop alone, the "tail" peptide, or a mixture of the two were at least 10-fold less potent inhibitors of clotting or protein C activation. A more constrained peptide analog was designed by deletion of an isoleucine within the C5-C6 disulfide loop, TM52-1 + 5C. This analog was a better inhibitor in both assay systems, having a Ki for protein C activation of 26 microM.