G domain dimerization controls dynamin's assembly-stimulated GTPase activity

Abstract

Dynamin is an atypical GTPase that catalyzes membrane fission during clathrin-mediated endocytosis. The mechanisms of dynamin’s basal and assembly-stimulated GTP hydrolysis are unknown, though both are indirectly influenced by the GTPase effector domain (GED). Here we present the 2.0Å resolution crystal structure of a minimal GTPase-GED fusion protein (GG) constructed from human dynamin 1, which has dimerized in the presence of the transition state mimic GDP.AlF4−. The structure reveals dynamin’s catalytic machinery and explains how assembly-stimulated GTP hydrolysis is achieved through G domain dimerization. A sodium ion present in the active site suggests that dynamin uses a cation to compensate for the developing negative charge in the transition state in the absence of an arginine finger. Structural comparison to the rat dynamin G domain reveals key conformational changes that promote G domain dimerization and stimulated hydrolysis. The structure of the GG dimer provides new insight into the mechanisms underlying dynamin-catalyzed membrane fission.

Dynamin 2 homozygous mutation in humans with a lethal congenital syndrome

“…[9][10][11] We observed an B20% reduction in patient fibroblasts with the p.Phe379Val mutation (Figure 2c). 10,11 As dynamin is a GTPase that can tubulate and sever membranes, 12,13 we measured the basal GTPase activity of wild-type and p.Phe379Val DNM2 recombinant proteins and found that the p.Phe379Val mutant's activity was reduced by 20% (Figure 2d). This mutant also failed to tubulate small unilammellar vesiscles in an in vitro assay (Figure 2e).…”

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…7B). GTPase -GTPase contacts, stabilized by GTP binding (Chappie et al 2010), generate a very different twofold relationship, which coupled with the stem-dimer would produce a repeating polymer (Fig. 7C).…”

Structural basis for the nucleotide-dependent dimerization of the large G protein atlastin-1/SPG3A

“…In addition, GTPases of this family often depend on nucleotide-dependent homodimerization to facilitate GTP hydrolysis rather than heterodimerization with a GTPase activating protein (2). Such a regulatory mechanism has been established for guanylate-binding protein (GBP) and dynamin (3,4).…”

“…In low-resolution models of dynamin, the middle domain and GED form a stalk-like structure that is involved in self-assembly (6,7). Furthermore, the GED and the C terminus of the G domain form a three-helix bundle, called the bundle signaling element (BSE), which modulates dynamin function (4,8). A recent crystal structure of a minimal G domain-GED fusion protein revealed dimeric G domains in a catalytically competent transition state that was proposed to play a role in the disassembly of dynamin coats from membrane tubes proceeding the fission event (4,9).…”

“…Furthermore, the GED and the C terminus of the G domain form a three-helix bundle, called the bundle signaling element (BSE), which modulates dynamin function (4,8). A recent crystal structure of a minimal G domain-GED fusion protein revealed dimeric G domains in a catalytically competent transition state that was proposed to play a role in the disassembly of dynamin coats from membrane tubes proceeding the fission event (4,9). Membrane binding and the GTPase of dynamin are further regulated by the pleckstrin homology (PH) domain, which is located at the tip of the stalk distal to the G domain and serves an autoregulatory function (7).…”