Enzymatic analysis of a rhomboid intramembrane protease implicates transmembrane helix 5 as the lateral substrate gate.
Intramembrane proteolysis is a core regulatory mechanism of cells that raises a biochemical paradox of how hydrolysis of peptide bonds is accomplished within the normally hydrophobic environment of the membrane. Recent high-resolution crystal structures have revealed that rhomboid proteases contain a catalytic serine recessed into the plane of the membrane, within a hydrophilic cavity that opens to the extracellular face, but protected laterally from membrane lipids by a ring of transmembrane segments. This architecture poses questions about how substrates enter the internal active site laterally from membrane lipid. Because structures are static glimpses of a dynamic enzyme, we have taken a structure-function approach analyzing >40 engineered variants to identify the gating mechanism used by rhomboid proteases. Importantly, our analyses were conducted with a substrate that we show is cleaved at two intramembrane sites within the previously defined Spitz substrate motif. Engineered mutants in the L1 loop and active-site region of the GlpG rhomboid protease suggest an important structural, rather than dynamic, gating function for the L1 loop that was first proposed to be the substrate gate. Conversely, three classes of mutations that promote transmembrane helix 5 displacement away from the protease core dramatically enhanced enzyme activity 4- to 10-fold. Our functional analyses have identified transmembrane helix 5 movement to gate lateral substrate entry as a rate-limiting step in intramembrane proteolysis. Moreover, our mutagenesis also underscores the importance of other residue interactions within the enzyme that warrant further scrutiny.
Baker
Rosanna P
RP
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 507 Preclinical Teaching Building, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Young
Keith
K
Feng
Liang
L
Shi
Yigong
Y
Urban
Sinisa
S
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D008565:Membrane Proteins; D050505:Mutant Proteins; D010450:Endopeptidases; D010447:Peptide Hydrolases