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Structure of neurolysin reveals a deep channel that limits substrate access.

ABSTRACT: The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 10 residues by the limited size of the active site cavity. Some structural elements of neurolysin, including a five-stranded beta-sheet and the two active site helices, are conserved with other metallopeptidases. The connecting loop regions of these elements, however, are much extended in neurolysin, and they, together with other open coil elements, line the active site cavity. These potentially flexible elements may account for the ability of the enzyme to cleave a variety of sequences.

SUBMITTER: Brown CK

PROVIDER: S-EPMC30618 | biostudies-literature | 2001 Mar

REPOSITORIES: biostudies-literature

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Structure of neurolysin reveals a deep channel that limits substrate access.

Brown C K CK Madauss K K Lian W W Beck M R MR Tolbert W D WD Rodgers D W DW

Proceedings of the National Academy of Sciences of the United States of America 20010306 6

The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 1 ...[more]

PMID: 11248043

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Project description:Results from our molecular-modelling and site-directed-mutagenesis studies of prostaglandin I(2) synthase (PGIS) have suggested that the large PGIS cytoplasmic domain is anchored to the endoplasmic reticulum (ER) membrane by the N-terminal segment in a way that orients the substrate access channel opening to face the membrane. To test this hypothesis we have explored the accessibility of the PGIS substrate channel opening to site-specific antibodies. The working three-dimensional PGIS model constructed by protein homology modelling was used to predict surface portions near the substrate access channel opening. Two peptides corresponding to the surface immediately near the opening [residues 66-75 (P66-75) and 95-116 (P95-116)], and two other peptides corresponding to the surface about 10-20 A (1 A=0.1 nm) away from the opening [residues 366-382 (P366-382) and 472-482 (P472-482)] were used to prepare site-specific antibodies. All four antipeptide antibodies specifically recognized the synthetic segments of human PGIS and recombinant PGIS, as shown by binding assays and Western-blot analysis. The site-specific antibodies were used to probe the accessibility of the substrate access channel opening in transiently transfected COS-1 cells expressing recombinant human PGIS, and in spontaneously transformed human endothelial cell line ECV cells expressing endogenous human PGIS. Immunofluorescence staining was performed for cells selectively permeabilized with streptolysin O and for cells whose membranes were permeabilized with detergent. Antibodies to peptides in the immediate vicinity of the substrate channel (P66-75 and P95-116) bound to their targets only after general permeabilization with Triton X-100. In contrast, the two antibodies to peptides further from the channel opening (P366-382 and P472-482) bound to their targets even in cells with intact ER membranes. These observations support our topology model in which the PGIS substrate access channel opening is positioned close to the ER membrane.

Project description:Cytochrome P450s (CYPs) are a family of enzymes responsible for metabolizing nearly 80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, which may result in improper dosing and severe adverse drug reactions. CYPs have low sequence conservation, making it difficult to anticipate whether variant effects measured in one CYP may extend to others based on sequence alone. Even closely related CYPs, like CYP2C9 and its closest homolog CYP2C19, have distinct phenotypic properties despite sharing 92% of their sequences. Thus, we used Variant Abundance by Massively Parallel sequencing (VAMP-seq) to measure the protein abundance of 7,660 missense variants in CYP2C19 expressed in cultured human cells. Our results confirmed positions and structural features critical for CYP function and revealed how variants at positions conserved across all eukaryotic CYPs influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core, and that substitutions in some regions reduced abundance in CYP2C19 but not CYP2C9. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. When nearby amino acids were exchanged in double and triple mutants, we found distinct interactions between the sites in CYP2C19 and CYP2C9, revealing a region that is partially responsible for the difference in thermodynamic stability between the two homologs. Since these positions are also important for determining substrate specificity, there may be an evolutionary tradeoff between stability and altered enzymatic function. Finally, we used our data to analyze 368 previously unannotated human variants, finding that 43% had decreased abundance. Thus, by comparing variant effects between two closely related, important human genes, we have uncovered regions underlying their functional differences and paved the way for a more complete understanding of one of the most versatile families of enzymes.

Dataset Information

Structure of neurolysin reveals a deep channel that limits substrate access.

Publications

Structure of neurolysin reveals a deep channel that limits substrate access.

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