Abstract: The protein Pex19p functions as a receptor and chaperone of peroxisomal membrane proteins (PMPs). The crystal structure of the folded C-terminal part of the receptor reveals a globular domain that displays a bundle of three long helices in an antiparallel arrangement. Complementary functional experiments, using a range of truncated Pex19p constructs, show that the structured alpha-helical domain binds PMP-targeting signal (mPTS) sequences with about 10 muM affinity. Removal of a conserved N-terminal helical segment from the mPTS recognition domain impairs the ability for mPTS binding, indicating that it forms part of the mPTS-binding site. Pex19p variants with mutations in the same sequence segment abolish correct cargo import. Our data indicate a divided N-terminal and C-terminal structural arrangement in Pex19p, which is reminiscent of a similar division in the Pex5p receptor, to allow separation of cargo-targeting signal recognition and additional functions.
Abstract: The crystal structure of the S189D+A226G rat chymotrypsin-B mutant has been determined at 2.2 angstroms resolution. This mutant is the most trypsin-like mutant so far in the line of chymotrypsin-to-trypsin conversions, aiming for a more complete understanding of the structural basis of substrate specificity in pancreatic serine proteases. A226G caused significant rearrangements relative to S189D chymotrypsin, allowing an internal conformation of Asp189 which is close to that in trypsin. Serious distortions remain, however, in the activation domain, including zymogen-like features. The pH-profile of activity suggests that the conformation of the S1-site of the mutant is influenced also by the P1 residue of the substrate.
Abstract: Folded and functional proteins destined for translocation from the cytosol into the peroxisomal matrix are recognized by two different peroxisomal import receptors, Pex5p and Pex7p. Both cargo-loaded receptors dock on the same translocon components, followed by cargo release and receptor recycling, as part of the complete translocation process. Recent structural and functional evidence on the Pex5p receptor has provided insight on the molecular requirements of specific cargo recognition, while the remaining processes still remain largely elusive. Comparison of experimental structures of Pex5p and a structural model of Pex7p reveal that both receptors are built by ring-like arrangements with cargo binding sites, central to the respective structures. Although, molecular insight into the complete peroxisomal translocon still remains to be determined, emerging data allow to deduce common molecular principles that may hold for other translocation systems as well.
Abstract: Atomic resolution (<or=1.2 A) serine protease intermediate structures revealed that the strength of the hydrogen bonds between the enzyme and the substrate changed during catalysis. The well-conserved hydrogen bonds of antiparallel beta-sheet between the enzyme and the substrate become significantly shorter in the transition from a Michaelis complex analogue (Pontastacus leptodactylus (narrow-fingered crayfish) trypsin (CFT) in complex with Schistocerca gregaria (desert locust) trypsin inhibitor (SGTI) at 1.2 A resolution) to an acyl-enzyme intermediate (N-acetyl-Asn-Pro-Ile acyl-enzyme intermediate of porcine pancreatic elastase at 0.95 A resolution) presumably synchronously with the nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. This is interpreted as an active mechanism that utilizes the energy released from the stronger hydrogen bonds to overcome the energetic barrier of the nucleophilic attack by the hydroxyl group of the catalytic serine. In the CFT:SGTI complex this hydrogen bond shortening may be hindered by the 27I-32I disulfide bridge and Asn-15I of SGTI. The position of the catalytic histidine changes slightly as it adapts to the different nucleophilic attacker during the transition from the Michaelis complex to the acyl-enzyme state, and simultaneously its interaction with Asp-102 and Ser-214 becomes stronger. The oxyanion hole hydrogen bonds provide additional stabilization for acyl-ester bond in the acyl-enzyme than for scissile peptide bond of the Michaelis complex. Significant deviation from planarity is not observed in the reactive bonds of either the Michaelis complex or the acyl-enzyme. In the Michaelis complex the electron distribution of the carbonyl bond is distorted toward the oxygen atom compared to other peptide bonds in the structure, which indicates the polarization effect of the oxyanion hole.
Abstract: We have previously shown that a trypsin inhibitor from desert locust Schistocerca gregaria (SGTI) is a taxon-specific inhibitor that inhibits arthropod trypsins, such as crayfish trypsin, five orders of magnitude more effectively than mammalian trypsins. Thermal denaturation experiments, presented here, confirm the inhibition kinetics studies; upon addition of SGTI the melting temperatures of crayfish and bovine trypsins increased 27 degrees C and 4.5 degrees C, respectively. To explore the structural features responsible for this taxon specificity we crystallized natural crayfish trypsin in complex with chemically synthesized SGTI. This is the first X-ray structure of an arthropod trypsin and also the highest resolution (1.2A) structure of a trypsin-protein inhibitor complex reported so far. Structural data show that in addition to the primary binding loop, residues P3-P3' of SGTI, the interactions between SGTI and the crayfish enzyme are also extended over the P12-P4 and P4'-P5' regions. This is partly due to a structural change of region P10-P4 in the SGTI structure induced by binding of the inhibitor to crayfish trypsin. The comparison of SGTI-crayfish trypsin and SGTI-bovine trypsin complexes by structure-based calculations revealed a significant interaction energy surplus for the SGTI-crayfish trypsin complex distributed over the entire binding region. The new regions that account for stronger and more specific binding of SGTI to crayfish than to bovine trypsin offer new inhibitor sites to engineer in order to develop efficient and specific protease inhibitors for practical use.
