Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics

Abstract

Rhomboid proteases reside within cellular membranes, but the advantage of this unusual environment is unclear. We discovered membrane immersion allows substrates to be identified in a fundamentally-different way, based initially upon exposing ‘masked’ conformational dynamics of transmembrane segments rather than sequence-specific binding. EPR and CD spectroscopy revealed that the membrane restrains rhomboid gate and substrate conformation to limit proteolysis. True substrates evolved intrinsically-unstable transmembrane helices that both become unstructured when not supported by the membrane, and facilitate partitioning into the hydrophilic, active-site environment. Accordingly, manipulating substrate and gate dynamics in living cells shifted cleavage sites in a manner incompatible with extended sequence binding, but correlated with a membrane-and-helix-exit propensity scale. Moreover, cleavage of diverse non-substrates was provoked by single-residue changes that destabilize transmembrane helices. Membrane immersion thus bestows rhomboid proteases with the ability to identify substrates primarily based on reading their intrinsic transmembrane dynamics.DOI: http://dx.doi.org/10.7554/eLife.00173.001

Features of Pro-σ K Important for Cleavage by SpoIVFB, an Intramembrane Metalloprotease

“…These enzymes normally require helix-destabilizing residues in the TMS that gets cleaved (42,62). Recently, studies of rhomboids reconstituted into proteoliposomes or expressed in cells indicated that helix-destabilizing residues in the target TMS of the substrate, as well as the dynamic properties of the enzyme in the membrane, play the primary role in substrate recognition (65).…”

Crystal Structures and Inhibition Kinetics Reveal a Two-Stage Catalytic Mechanism with Drug Design Implications for Rhomboid Proteolysis

“…Therefore, to gain a better understanding of the mechanism underlying intramembrane catalysis, we sought to adapt this approach to characterize the inhibition kinetics of substrate-mimicking peptide aldehydes. Next, since mounting evidence indicates that cell membranes affect the properties of rhomboid proteases beyond just serving as their environment (Bondar et al, 2009; Moin and Urban, 2012; Urban and Moin, 2014; Vinothkumar, 2011), we developed conditions to crystallize a catalytically active rhomboid protease in a membrane. Soaking rhomboid crystallized from bicelles with the most potent substrate peptide aldehydes produced high-resolution structures that revealed the characteristics of substrate stabilization during catalysis by a membrane-immersed rhomboid protease.…”

Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain

“…Recent work on rhomboid proteases has demonstrated that this family of I-CLiPs achieves substrate specificity via a mechanism that is dependent on the transmembrane dynamics of the substrate rather than its sequence of amino acids (6,7). Here, rhomboid possesses a very weak binding affinity for substrate and, in a rate-driven reaction, only cleaves those substrates that have unstable TMD helices that have had time to unfold into the catalytic active site, where they are cleaved before they can dissociate from the enzyme-substrate complex.…”