title: Networks of Dynamic Allostery Regulate Enzyme Function.
abstract: Many protein systems rely on coupled dynamic networks to allosterically regulate function. However, the broad conformational space sampled by non-coherently dynamic systems has precluded detailed analysis of their communication mechanisms. Here, we have developed a methodology that combines the high sensitivity afforded by nuclear magnetic resonance relaxation techniques and single-site multiple mutations, termed RASSMM, to identify two allosterically coupled dynamic networks within the non-coherently dynamic enzyme cyclophilin A. Using this methodology, we discovered two key hotspot residues, Val6 and Val29, that communicate through these networks, the mutation of which altered active-site dynamics, modulating enzymatic turnover of multiple substrates. Finally, we utilized molecular dynamics simulations to identify the mechanism by which one of these hotspots is coupled to the larger dynamic networks. These studies confirm a link between enzyme dynamics and the catalytic cycle of cyclophilin A and demonstrate how dynamic allostery may be engineered to tune enzyme function.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, 12801 East 17th Avenue, MS 8101, Aurora, CO 80045, USA.
authors.forename: Michael Joseph
authors.initials: MJ
authors.lastname: Holliday
authors.affiliation: Department of Chemistry, Institute for Advanced Study, Technische Universität München, 85748 Garching, Germany.
authors.forename: Carlo
authors.initials: C
authors.lastname: Camilloni
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
authors.forename: Geoffrey Stuart
authors.initials: GS
authors.lastname: Armstrong
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
authors.forename: Michele
authors.initials: M
authors.lastname: Vendruscolo
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, 12801 East 17th Avenue, MS 8101, Aurora, CO 80045, USA. Electronic address: elan.eisenmesser@ucdenver.edu.
authors.forename: Elan Zohar
authors.initials: EZ
authors.lastname: Eisenmesser
mesh_terms: D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D055162:Biocatalysis; D020134:Catalytic Domain; D021984:Cyclophilin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D056004:Molecular Dynamics Simulation; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014633:Valine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D021984:Cyclophilin A; D014633:Valine
keywords: allostery; cyclophilin A; dynamics; isomerization; nuclear magnetic resonance; protein engineering