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CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin
Hemocyanins are multimeric oxygen transport proteins present in the blood of arthropods and molluscs, containing up to 8 oxygen-binding functional units per monomer. In molluscs, hemocyanins are assembled in decamer ‘building blocks’ formed of 5 dimer ‘plates’, routinely forming didecamer or higher-...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10286996/ https://www.ncbi.nlm.nih.gov/pubmed/37347755 http://dx.doi.org/10.1371/journal.pone.0287294 |
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author | Pasqualetto, Gaia Mack, Andrew Lewis, Emily Cooper, Ryan Holland, Alistair Borucu, Ufuk Mantell, Judith Davies, Tom Weckener, Miriam Clare, Dan Green, Tom Kille, Pete Muhlhozl, Alex Young, Mark T. |
author_facet | Pasqualetto, Gaia Mack, Andrew Lewis, Emily Cooper, Ryan Holland, Alistair Borucu, Ufuk Mantell, Judith Davies, Tom Weckener, Miriam Clare, Dan Green, Tom Kille, Pete Muhlhozl, Alex Young, Mark T. |
author_sort | Pasqualetto, Gaia |
collection | PubMed |
description | Hemocyanins are multimeric oxygen transport proteins present in the blood of arthropods and molluscs, containing up to 8 oxygen-binding functional units per monomer. In molluscs, hemocyanins are assembled in decamer ‘building blocks’ formed of 5 dimer ‘plates’, routinely forming didecamer or higher-order assemblies with d5 or c5 symmetry. Here we describe the cryoEM structures of the didecamer (20-mer) and tridecamer (30-mer) forms of a novel hemocyanin from the slipper limpet Crepidula fornicata (SLH) at 7.0 and 4.7 Å resolution respectively. We show that two decamers assemble in a ‘tail-tail’ configuration, forming a partially capped cylinder, with an additional decamer adding on in ‘head-tail’ configuration to make the tridecamer. Analysis of SLH samples shows substantial heterogeneity, suggesting the presence of many higher-order multimers including tetra- and pentadecamers, formed by successive addition of decamers in head-tail configuration. Retrieval of sequence data for a full-length isoform of SLH enabled the use of Alphafold to produce a molecular model of SLH, which indicated the formation of dimer slabs with high similarity to those found in keyhole limpet hemocyanin. The fit of the molecular model to the cryoEM density was excellent, showing an overall structure where the final two functional units of the subunit (FU-g and FU-h) form the partial cap at one end of the decamer, and permitting analysis of the subunit interfaces governing the assembly of tail-tail and head-tail decamer interactions as well as potential sites for N-glycosylation. Our work contributes to the understanding of higher-order oligomer formation in molluscan hemocyanins and demonstrates the utility of Alphafold for building accurate structural models of large oligomeric proteins. |
format | Online Article Text |
id | pubmed-10286996 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-102869962023-06-23 CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin Pasqualetto, Gaia Mack, Andrew Lewis, Emily Cooper, Ryan Holland, Alistair Borucu, Ufuk Mantell, Judith Davies, Tom Weckener, Miriam Clare, Dan Green, Tom Kille, Pete Muhlhozl, Alex Young, Mark T. PLoS One Research Article Hemocyanins are multimeric oxygen transport proteins present in the blood of arthropods and molluscs, containing up to 8 oxygen-binding functional units per monomer. In molluscs, hemocyanins are assembled in decamer ‘building blocks’ formed of 5 dimer ‘plates’, routinely forming didecamer or higher-order assemblies with d5 or c5 symmetry. Here we describe the cryoEM structures of the didecamer (20-mer) and tridecamer (30-mer) forms of a novel hemocyanin from the slipper limpet Crepidula fornicata (SLH) at 7.0 and 4.7 Å resolution respectively. We show that two decamers assemble in a ‘tail-tail’ configuration, forming a partially capped cylinder, with an additional decamer adding on in ‘head-tail’ configuration to make the tridecamer. Analysis of SLH samples shows substantial heterogeneity, suggesting the presence of many higher-order multimers including tetra- and pentadecamers, formed by successive addition of decamers in head-tail configuration. Retrieval of sequence data for a full-length isoform of SLH enabled the use of Alphafold to produce a molecular model of SLH, which indicated the formation of dimer slabs with high similarity to those found in keyhole limpet hemocyanin. The fit of the molecular model to the cryoEM density was excellent, showing an overall structure where the final two functional units of the subunit (FU-g and FU-h) form the partial cap at one end of the decamer, and permitting analysis of the subunit interfaces governing the assembly of tail-tail and head-tail decamer interactions as well as potential sites for N-glycosylation. Our work contributes to the understanding of higher-order oligomer formation in molluscan hemocyanins and demonstrates the utility of Alphafold for building accurate structural models of large oligomeric proteins. Public Library of Science 2023-06-22 /pmc/articles/PMC10286996/ /pubmed/37347755 http://dx.doi.org/10.1371/journal.pone.0287294 Text en © 2023 Pasqualetto et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Pasqualetto, Gaia Mack, Andrew Lewis, Emily Cooper, Ryan Holland, Alistair Borucu, Ufuk Mantell, Judith Davies, Tom Weckener, Miriam Clare, Dan Green, Tom Kille, Pete Muhlhozl, Alex Young, Mark T. CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin |
title | CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin |
title_full | CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin |
title_fullStr | CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin |
title_full_unstemmed | CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin |
title_short | CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin |
title_sort | cryoem structure and alphafold molecular modelling of a novel molluscan hemocyanin |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10286996/ https://www.ncbi.nlm.nih.gov/pubmed/37347755 http://dx.doi.org/10.1371/journal.pone.0287294 |
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