Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia co ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation2CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP306943/2015-8; 420567/2016-099999.004913/2015-092015/15822-1; 2012/01953-9; 2016/05019-0; 2012/50161-8Precision Medicine Initiative (PRiME) at the University of Toronto internal fellowship [PMRF2019-007]; Canadian Institutes of Health Research (CIHR) postdoctoral fellowshipCanadian Institutes of Health Research (CIHR); CNPq-Brazil fellowship [202192/2015-6]; Saskatchewan Health Research Foundation postdoctoral fellowship; Ontario Graduate Scholarship (OGS)Ontario Graduate Scholarship; Department of Biochemistry at the University of Toronto; Centre for Pharmaceutical Oncology (University of Toronto); CIHR Training Program in Protein Folding and Interaction Dynamics: Principles and Diseases fellowshipCanadian Institutes of Health Research (CIHR) [TGF-53910]; University of Toronto Fellowship from the Department of Biochemistry; OGS fellowship; NSERC PGS-D2 fellowship; CIHR Emerging Team Grants from the Institute of Infection and ImmunityCanadian Institutes of Health Research (CIHR) [XNE-86945]; CIHR Project grantCanadian Institutes of Health Research (CIHR) [PJT-148564]; Global Affairs Canada (Canada); CAPES (Brazil)CAPES [99999.004913/2015-09]; NSERCNatural Sciences and Engineering Research Council of Canada [RGPIN-2015-04877, DG-20234]; Canada Research Chairs ProgramCanada Research Chairs; CIHR new investigator programCanadian Institutes of Health Research (CIHR); FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2015/15822-1, 2012/01953-9, 2016/05019-0, 2012/50161-8]; CNPqNational Council for Scientific and Technological Development (CNPq) [306943/2015-8, 420567/2016-0]; AbbVie [1097737]; BayerBayer AG [1097737]; Boehringer IngelheimBoehringer Ingelheim [1097737]; Genome Canada through Ontario Genomics Institute GrantGenome Canada [1097737, OGI-055]; GlaxoSmithKlineGlaxoSmithKline [1097737]; JanssenJohnson & Johnson USAJanssen Biotech Inc [1097737]; Lilly CanadaEli Lilly [1097737]; MerckMerck & Company [1097737]; Novartis Research Foundation [1097737]; Ontario Ministry of Economic Development and Innovation [1097737]; PfizerPfizer [1097737]; TakedaTakeda Pharmaceutical Company Ltd [1097737]; Wellcome Trust GrantWellcome Trust [1097737, 092809/Z/10/Z]; Canada Foundation for InnovationCanada Foundation for Innovation; NSERCNatural Sciences and Engineering Research Council of Canada; University of Saskatchewan; Government of Saskatchewan; Western Economic Diversification Canada; National Research Council Canada; CIHRCanadian Institutes of Health Research (CIHR
