p190RhoGAP proteins contain pseudoGTPase domains

Abstract

The two p190RhoGAP proteins, p190RhoGAP-A and -B, are key regulators of Rho GTPase signaling and are essential for actin cytoskeletal structure and contractility. Here we report the discovery of two evolutionarily conserved GTPase-like domains located in the ‘middle domain’, previously thought to be unstructured. Deletion of these domains reduces RhoGAP activity. Crystal structures, MANT-GTPγS binding, thermal denaturation, biochemical assays and sequence homology analysis all strongly support defects in nucleotide-binding activity. Analysis of p190RhoGAP proteins therefore indicates the presence of two previously unidentified domains which represent an emerging group of pseudoenzymes, the pseudoGTPases.

Synchrotron Big Data Science

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PseudoGTPase domains in p190RhoGAP proteins: a mini-review

“…Pseudoenzymes have been described in the protein kinase, phosphatase, protease, GTPase and other families [3, 5], and are thought to encompass approximately 10% of each enzyme fold [2, 6]. For the small GTPase family a number of pseudoGTPases have been identified over the last four or five years [7–9]. Consequently there is a growing group of annotated pseudoGTPases within the fold.…”

“…Consequently there is a growing group of annotated pseudoGTPases within the fold. We recently identified multiple pseudoGTPase domains in the p190RhoGAP proteins, which are important regulators of RhoA signaling [9, 10]. In this mini-review we discuss the discovery and characterization of these multiple pseudoGTPase domains and summarize the current state of the pseudoGTPase group using the p190RhoGAP proteins as examples.…”

“…These cryptic GTPase domains can be elusive in sequence-based bioinformatics searches, but upon crystal structure determination have been revealed to adopt the canonical GTPase-like fold. Cryptic GTPase domains have been found in CENP-M, fungal dynein LIC, and the p190RhoGAP proteins [7–9], and their collective identification raised the possibility that a larger number of cryptic pseudoGTPases have yet to be identified. Consequently, similar to the pseudokinases, the pseudoGTPases contain catalytically inactive (e.g.…”

“…This region of approximately 700 residues had not, however, been fully analyzed since the late 1990s. We conducted sequence analysis and discovered an extensive region of strongly predicted secondary structure encompassing approximately 400 residues [9]. Secondary structure-based fold homology detection suggested that this region contained two GTPase-like domains which we termed pG1 and pG2, but also indicated low sequence identity of less than 20% compared to any small GTPase domain.…”