Abstract
Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5′-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains.
Highlights
Members of the dynamin superfamily comprise a family of conserved GTPases, which are mostly found in the eukaryotic kingdom and mediate functions typically related to membrane remodeling [1,2]
The stalk (S), which participates in higher-order oligomerization, and the B-insert were replaced by a GSGSGSGS linker, which continued with the C-terminal helix of the bundle signaling element (BSE) (Fig. 1A) [38]
Both crystals diffracted to 2.3 Aresolution, and the structures were solved by molecular replacement using the GG construct of human dynamin-1 as a search model (Tables 1, 2)
Summary
Members of the dynamin superfamily comprise a family of conserved GTPases, which are mostly found in the eukaryotic kingdom and mediate functions typically related to membrane remodeling [1,2]. The GTPase domains of most dynamin superfamily members contain five conserved GTP-binding motifs (G1-5), similar to small Ras-like GTPases [3]. A conserved threonine in switch-I (G2) and the conserved residues DxxG of switch-II (G3) are involved in Mg2+ binding and GTP (Guanosine-59-triphosphate) hydrolysis. These regions are rather flexible in the GDP-bound form but are stabilized in GTP-bound state [5]. The nucleotide binding affinity of dynamins is typically low, with specificity for GTP provided by the mostly conserved N/TKxD motif (G4) [2,3]. The G5-motif is involved in binding the ribose moiety
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