An internal water-retention site in the rhomboid intramembrane protease GlpG ensures catalytic efficiency.
Rhomboid proteases regulate key cellular pathways, but their biochemical mechanism including how water is made available to the membrane-immersed active site remains ambiguous. We performed four prolonged molecular dynamics simulations initiated from both gate-open and gate-closed states of Escherichia coli rhomboid GlpG in a phospholipid bilayer. GlpG was notably stable in both gating states, experiencing similar tilt and local membrane thinning, with no observable gating transitions, highlighting that gating is rate-limiting. Analysis of dynamics revealed rapid loss of crystallographic waters from the active site, but retention of a water cluster within a site formed by His141, Ser181, Ser185, and/or Gln189. Experimental interrogation of 14 engineered mutants revealed an essential role for at least Gln189 and Ser185 in catalysis with no effect on structural stability. Our studies indicate that spontaneous water supply to the intramembrane active site of rhomboid proteases is rare, but its availability for catalysis is ensured by an unanticipated active site element, the water-retention site.
Department of Chemistry, New York University, New York, NY 10003, USA.
Yanzi
Y
Zhou
Syed M
SM
Moin
Sinisa
S
Urban
Yingkai
Y
Zhang
D055162:Biocatalysis; D020134:Catalytic Domain; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000737:chemistry* / Q000378:metabolism
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D008051:Lipid Bilayers; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014867:Water; D010450:Endopeptidases