Structure-based analysis of the guanine nucleotide exchange factor SmgGDS reveals armadillo-repeat motifs and key regions for activity and GTPase binding

Abstract

Small GTPases are molecular switches that have critical biological roles and are controlled by GTPase-activating proteins and guanine nucleotide exchange factors (GEFs). The smg GDP dissociation stimulator (SmgGDS) protein functions as a GEF for the RhoA and RhoC small GTPases. SmgGDS has various regulatory roles, including small GTPase trafficking and localization and as a molecular chaperone, and interacts with many small GTPases possessing polybasic regions. Two SmgGDS splice variants, SmgGDS-558 and SmgGDS-607, differ in GEF activity and binding affinity for RhoA depending on the lipidation state, but the reasons for these differences are unclear. Here we determined the crystal structure of SmgGDS-558, revealing a fold containing tandem copies of armadillo repeats not present in other GEFs. We also observed that SmgGDS harbors distinct positively and negatively charged regions, both of which play critical roles in binding to RhoA and GEF activity. This is the first report demonstrating a relationship between the molecular function and atomic structure of SmgGDS. Our findings indicate that the two SmgGDS isoforms differ in GTPase binding and GEF activity, depending on the lipidation state, thus providing useful information about the cellular functions of SmgGDS in cells.

GEF mechanism revealed by the structure of SmgGDS-558 and farnesylated RhoA complex and its implication for a chaperone mechanism

“…S1A and Table S1). SmgGDS/ RhoA forms a 1:1 complex in solution revealed by small angle X-ray scattering, multiangle light scattering, and gel-filtration analyses (19). The N-terminal 10 residues (amino acids 77-86) and C-terminal 3 residues (amino acids 556-558) of SmgGDS-558 were disordered.…”

“…In complexes II and III, the electron density of RhoA PBR (amino acids 181-189) was observed, but the density was so poor that only main-chain atoms were modeled. In addition, the P loop and its flanking region (amino acids [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] were disordered in complexes II and III ( Fig. 1 B-D and SI Appendix, Fig.…”

“…SmgGDS-558 prefers prenylated RhoA, whereas SmgGDS-607 prefers nonprenylated RhoA. Recently, we reported that these splice variants differ in GEF activity and binding affinity for RhoA, depending on their prenylation state (19). We also determined the crystal structure of SmgGDS-558 and revealed that the positively charged concave surface of SmgGDS-558 is a key region for GEF activity (19).…”

“…Recently, we reported that these splice variants differ in GEF activity and binding affinity for RhoA, depending on their prenylation state (19). We also determined the crystal structure of SmgGDS-558 and revealed that the positively charged concave surface of SmgGDS-558 is a key region for GEF activity (19). However, the precise mechanism underlying how SmgGDS stimulates GDP/GTP exchange of RhoA and how SmgGDS isoforms can distinguish RhoA prenylation remain unclear.…”

Functions for Cdc42p BEM Adaptors in Regulating a Differentiation-Type MAP Kinase Pathway

“…This region exhibits sequence similarity to members of the SmgGDS family of proteins. SmgGDS proteins contain Armadillo (ARM) repeats ( 86, 87 ) that mediate interactions with Rho GTPases ( 86, 88–92 ) and other proteins. The predicted structure of Bem4p was determined by bioinformatics algorithms Phyre 2 and iTASSER, which generated similar structures with >90% confidence and theoretical root mean square deviation (iRMSD) values above cutoff.…”