PROBING THE STRUCTURAL DYNAMICS OF THE ENZYME-SUBSTRATE COMPLEX FOR INTRAMEMBRANE-CLEAVING

The Intramembrane-Cleaving Proteases (I-CLiPs) serve to cleave single span transmembrane TM proteins within the membrane interior in order to release soluble effectors for signaling (regulated intramembrane proteolysis) or help degrade mis-folded or no longer needed membrane proteins.  They are poorly understood at biochemically and biophysically.  A good anecdote to this point is that the E. Coli enzyme, GlpG, has been solved at atomic resolution many fold over, but its cellular role and physiological substrates are wholly unknown.  Of course, these enzymes also have a dark side, biologically speaking, in that promiscuity location-wise of the cleavage event of the Alzheimer’s precursor protein yields the infamous “toxic” forms of the Ab peptide fragments known to be involved in Alzheimer’s disease.  To address the events of substrate recognition and cleavage, we have employed a newly developed strategy combining isotopic labeling and exchange with deep-UV excited resonance Raman (dUVRR).  DUVRR is sensitive to hydration status in the amide I mode and also to Psi angle distribution in the well-resolved amide III modes.  The deuteration and isotopic labeling of the enzyme and deuterium exchange of the soluble portion of the substrates peptide backbones enables direct visualization of the unlabeled substrate TM a-helix structure(s). DUVRR spectra (197 nm excitation) of fully ¹⁵N,¹³C, ²H-labeled I-CLiP enzyme interacting with the cleavable Gurken protein in D₂O revealed a significant change in the Psi angle dependent amide III mode, indicating helical unwinding. Additional changes in the hydration sensitive amide I mode and aromatic modes further reveal extensive hydration changes.  Interestingly these events are not contiguous in time with one another during the time-course of the assay, implying cleavage (requiring water for catalysis) and unwinding are not directly correlated with one another during the experiment.  Further, similar experiments utilizing mutants in the enzymes catalytic residues, only displayed helical unwinding, but lacked significant hydration changes. Overall these results imply that binding of the substrate and enzyme result in unwinding to some extent of the TM a-helix, while extensive hydration is predicated by the ability to carry out cleavage events.  This implies that residence time of the unwound substrate in the active site itself may be a key determinant of efficient cleavage.