{"doi":"10.1002/pro.70287","title":"Hydrophobic residues in the D‐domain of a plasminogen‐binding M‐protein modulate α‐helicity, oligomerization, and surface anchoring in <i>Streptococcus pyogenes</i>","abstract":"Abstract Human plasminogen (hPg) binding M‐protein (PAM) is a major virulence determinant of Group A Streptococcus (GAS). PAM contains irregularly spaced heptad repeats, particularly within its C‐ and D‐domains. Our recent cryo‐EM structure of PAM from GAS strain AP53 (PAM AP53 ) revealed that these repeats do not favor coiled‐coil formation, but instead adopt triple‐helix bundle and helix–loop–helix motifs in the C‐ and D‐domains, respectively. Interactions between loops connecting helices in these domains maintain the compact conformation of PAM AP53 , correctly orienting human plasminogen (hPg) for activation by the co‐expressed streptokinase variant, SK2b. Despite the D‐domain being conserved across M‐proteins, its specific structural and functional roles remain unknown. To investigate the role of the D‐domain in PAM structure–function, we engineered three structure‐guided PAM AP53 variants, replacing nine heptad D‐domain residues with glycine (9G), alanine (9A), or leucine (9L). At 25°C, wild‐type (WT)‐PAM AP53 exists as a concentration‐ and temperature‐dependent mixture of dimers, tetramers, and higher‐order oligomers, which dissociate at physiological temperature. In contrast, PAM AP53 ‐9A and PAM AP53 ‐9L form thermostable oligomers while helix‐destabilizing PAM AP53 ‐9G exists as a monomer exhibiting structural loss at all temperatures. All variants retained high hPg‐binding affinity. However, unlike WT‐PAM AP53 , PAM AP53 ‐9A, and PAM AP53 ‐9L, PAM AP53 ‐9G poorly stimulated hPg activation. Moreover, whereas WT‐PAM AP53 , PAM AP53 ‐9A, and PAM AP53 ‐9L were covalently attached to the bacterial cell wall and displayed on the cell surface, PAM AP53 ‐9G, with a significantly reduced helical content, was not exposed on the bacterial surface. Our results reveal the importance of the D‐domain to secondary/quaternary structure, attachment of PAM AP53 to the GAS surface, and SK2b‐mediated activation of hPg.","journal":"Protein Science","year":2025,"id":554135,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9603,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":425919,"name":"Yetunde A. Ayinuola","orcid":"0000-0001-7803-8058","position":1,"is_corresponding":false},{"id":393197,"name":"Zhong Liang","orcid":"0000-0002-9392-635X","position":2,"is_corresponding":false},{"id":393200,"name":"Francis Castellino","orcid":"0000-0003-0317-9539","position":3,"is_corresponding":false},{"id":444533,"name":"Olawole Ayinuola","orcid":null,"position":0,"is_corresponding":true}],"reference_count":53,"raw_metadata":null,"created_at":"2026-07-19T02:54:45.872391Z","pmid":"40944438","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}