Efficient GTP hydrolysis requires 1) the correct positioning of a water molecule for a nucleophilic attack on the γ-phosphate, 2) neutralization of a negative charge that develops between the β- and γ-phosphates in the transition state, and 3) stabilization of the conformationally flexible switch regions within the catalytic core23,24
. These conditions are achieved either through interaction with a GTPase-activating protein (GAP)25,26
or through dimerization27
. The GG dimer structure reveals how dynamin accomplishes these requirements.
In the active site of each GG monomer, the main chain carbonyl of T65 and the backbone nitrogen of G139 position the catalytic water in line with the AlF4−
moiety (). A second water molecule further orients the catalytic water by acting as a bridge to the Q40 side chain and the G139 carbonyl oxygen. This type of indirect positioning has thus far only been observed in the tRNA modifying MnmE GTPase28
. D180 of the trans
stabilizing loop reaches across the dimer interface and forms hydrogen bonds with Q40, S41, and the main chain nitrogen of G62 (). These interactions stabilize the P-loop and switch I conformations such that they are poised for catalysis.
Catalytic machinery involved in dynamin GTP hydrolysis
Notably absent from the GG active site is a charge-compensating arginine finger. Instead, we observe additional electron density that we interpreted as a sodium ion (Supplementary Fig. 3a
). The ligands for this ion include the aluminum fluoride, b-phosphate, carbonyl oxygens of G60 and G62 in switch I, and S41 side chain in the P-loop (). This pattern of coordination is similar to that of the in cis
arginine finger of human guanylate binding protein 1 (hGBP1)29
() and the bound potassium ion in the active site of MnmE (), which mimics the function of an arginine finger28
. We hypothesized that the sodium in our GG structure serves a similar purpose and indeed found that GTP hydrolysis in both GG (Supplementary Fig. 3b
) and full-length dynamin (data not shown) is sensitive to the presence of different monovalent cations. Potassium, which is likely more physiologically relevant due to its higher intracellular concentration, induces a highesr hydrolysis rate than sodium, while rubidium and cesium impair turnover by increasing degrees.
The bound magnesium ion and K44 side chain also form hydrogen bonds with the β-phosphate and the aluminum fluoride (). These interactions mirror the effects of the sodium ion from the other side of the nucleotide and act in concert to stabilize the developing charge in the transition state. This explains the strong dominant-negative effects of dynamin(K44A), which is defective in both GTP binding and hydrolysis5,6
The preponderance of backbone interactions in stabilizing the bridging and catalytic waters (T65O
) and the bound cation (G60O
) explains inabilities to define dynamin’s basal catalytic mechanism by mutagenesis alone7,21,30
. Previous studies7,30
have nonetheless identified S45, T65, T141, and K142 as important functional residues. Defects associated with these side chains can now be rationalized by our GG structure. S45 and T65 coordinate the bound magnesium, the disruption of which would prevent proper nucleotide binding and charge compensation. T141 in contrast does not actively contribute to the GG active site or the dimer interface, despite its location in switch II. However, substitutions at this position could sterically interfere with the cis
stabilizing loop interactions that restrict the conformational flexibility of switch II. Indeed, T141Q and T141D mutations have much stronger defects in vitro
and in vivo
than a smaller T141A substitution7,30
. K142A moderately impairs dynamin GTP hydrolysis but strongly inhibits endocytosis30
. K142 can hydrogen bond with E153 at the base of the GG dimer (). Removal of this interaction could preferentially alter the dimerization-dependent functions of dynamin.