Insights into Substrate Gating in H. influenzae Rhomboid

Brooks, Cory L.; Lazareno-Saez, Christelle; Lamoureux, Jason S.; Mak, Michelle; Lemieux, M. Joanne

doi:10.1016/j.jmb.2011.01.046

Cited by 32 publications

(43 citation statements)

References 49 publications

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“…On the basis of the DFP structure, we constructed a model of peptide substrate bound at the GlpG active site after the nucleophilic attack of Ser-201 from the si-face of the scissile peptide bond (Fig. 5B) (27,36). The model is similar to the one proposed previously (37) and contains a turn at the scissile bond, which projects the N terminus of the substrate up and toward the aqueous solution.…”

Section: Discussionmentioning

confidence: 85%

“…It is interesting that some mutations introduced into the interface between TM helices S2 and S5 (e.g. F153A/W236A) enhanced the proteolytic activity of GlpG (34,36). The proximity of the mutations to a region that we now know may undergo side chain rearrangement suggests that their effects on activity do not have to be related to the hypothesized lateral movement of an entire helix (34).…”

Section: Unlike DCI Dfp Causes Irreversible Inhibition Of Rhomboidmentioning

confidence: 99%

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Catalytic Mechanism of Rhomboid Protease GlpG Probed by 3,4-Dichloroisocoumarin and Diisopropyl Fluorophosphonate

Xue

2012

Journal of Biological Chemistry

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Background:The rhomboid protease GlpG is a prototype of the serine intramembrane protease. Results: The crystal structure of GlpG in complex with diisopropyl fluorophosphonate was solved at 2.3 Å resolution. Conclusion:The crystal structure provides a model for the tetrahedral transitional state and reveals conformational changes within the active site. Significance: Learning how GlpG conducts catalysis is crucial for understanding the mechanism of intramembrane proteolysis by rhomboid proteases.

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Section: Discussionmentioning

confidence: 85%

Section: Unlike DCI Dfp Causes Irreversible Inhibition Of Rhomboidmentioning

confidence: 99%

Catalytic Mechanism of Rhomboid Protease GlpG Probed by 3,4-Dichloroisocoumarin and Diisopropyl Fluorophosphonate

Xue

2012

Journal of Biological Chemistry

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“…Crystal structures of the Escherichia coli rhomboid GlpG have shown that these residues are in close enough proximity to form a hydrogen bond (16,17). The attack onto the scissile bond of the substrate is proposed to occur at the si-face, opposite that of most other serine proteases (18,19). Another difference between rhomboids and classical serine proteases is the form in which they are translated.…”

mentioning

confidence: 99%

Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

Vosyka

Vinothkumar

Wolf

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S′ subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design.MALDI screening | covalent inhibition | regulated intramembrane proteolysis P roteolysis controls many important biological processes, such as apoptosis, antigen presentation, and blood coagulation. Selective digestion of protein substrates is possible by a combination of tight posttranslational control of protease activity (1) and the protease's substrate specificity, which generally is governed by the primary sequence around the scissile bond (2). The use of inhibitors and activity-based probes (ABPs) has led to a tremendous gain in understanding the roles of proteases within physiological and pathological processes (3). ABPs are small molecules that bind only to active enzymes, but not to zymogen or inhibitor-bound forms (4). ABPs generally consist of a detection tag, a spacer, and a "warhead." The warhead covalently binds to the target enzyme(s) and often is derived from a mechanism-based inhibitor. In the past, ABPs were used to study the activation, localization, and function of soluble proteases in a variety of organisms and disease models (5).Most proteases are soluble and surrounded by an aqueous environment. However, several families of intramembrane proteases exist (6-8): the metalloprotease family M50 (site-2 protease), the aspartic protease family A22 (signal peptide peptidase and γ-secretase), and the serine protease family S54 [rhomboid; numbering according to the MEROPS database (9)]. Rhomboid was discovered in 2001 as a protease in the EGF receptor signaling pathway in the fruitfly Drosophila melanogaster (10). Interestingly, rhomboid genes occur in all kingdoms of nature and are found in most sequenced organisms (11,12). Rhomboids appear to have a wide range of physiological functions, including bacterial protein export (13) and invasion by apicomplexan parasites (14,15), but the roles of many rhomboids remain to be discovered.Rhomboids catalyze peptide bond hydrolysis using a catalytic dyad formed by a serine residue in transmembrane domain...

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“…Another conserved motif in TMH6 (GxxxG) allows tight packing between the two TMHs that harbor the catalytic S-H dyad. Notably, the active site is accessible to water required for nucleophilic cleavage through a cleft facing the extracellular region that is capped by loop L5 [20, 21, 25–27]. Several atoms have been proposed to contribute to an oxyanion hole that stabilizes the developing negative charge on the carbonyl oxygen of the scissile bond during catalysis, including the main chain amide of the catalytic S and the side chain amides of two conserved TMH2 sidechains from the motif HxxxN (Figure 3A, shown in gray stick).…”

Section: Rhomboid Protease Structure and Mechanismmentioning

confidence: 99%

Bioinformatics perspective on rhomboid intramembrane protease evolution and function

Kinch

Grishin²

2013

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Endopeptidase classification based on catalytic mechanism and evolutionary history has proven to be invaluable to the study of proteolytic enzymes. Such general mechanistic- and evolutionary- based groupings have launched experimental investigations, because knowledge gained for one family member tends to apply to the other closely related enzymes. The serine endopeptidases represent one of the most abundant and diverse groups, with their apparently successful proteolytic mechanism having arisen independently many times throughout evolution, giving rise to the well-studied soluble chemotrypsins and subtilisins, among many others. A large and diverse family of polytopic transmembrane proteins known as rhomboids has also evolved the serine protease mechanism. While the spatial structure, mechanism, and biochemical function of this family as intramembrane proteases has been established, the cellular roles of these enzymes as well as their natural substrates remain largely undetermined. While the evolutionary history of rhomboid proteases has been debated, sorting out the relationships among current day representatives should provide a solid basis for narrowing the knowledge gap between their biochemical and cellular functions. Indeed, some functional characteristics of rhomboid proteases can be gleaned from their evolutionary relationships. Finally, a specific case where phylogenetic profile analysis has identified proteins that contain a C-terminal processing motif (GlyGly-Cterm) as co-occurring with a set of bacterial rhomboid proteases provides an example of potential target identification through bioinformatics.

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Insights into Substrate Gating in H. influenzae Rhomboid

Cited by 32 publications

References 49 publications

Catalytic Mechanism of Rhomboid Protease GlpG Probed by 3,4-Dichloroisocoumarin and Diisopropyl Fluorophosphonate

Catalytic Mechanism of Rhomboid Protease GlpG Probed by 3,4-Dichloroisocoumarin and Diisopropyl Fluorophosphonate

Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

Bioinformatics perspective on rhomboid intramembrane protease evolution and function

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