Mutational Analysis of the Arf1•GTP/Arf GAP Interface Reveals an Arf1 Mutant that Selectively Affects the Arf GAP ASAP1

SummaryArf1 is a GTP binding protein that functions at a number of cellular sites to control membrane traffic and actin remodeling. Arf1 is regulated by site-specific GTPase-activating proteins (GAPs). The combined results of crystallographic and biochemical studies [1–3] have led to the proposal that Arf1 GAPs differ in the specific interface formed with Arf1. To test this hypothesis, we have used mutagenesis to examine the interaction of three Arf GAPs (ASAP1, AGAP1, and ArfGAP1) with switch 1, switch 2, and α helix3 of Arf1. The GAPs were similar in being affected by mutations in switch 1 and 2. However, effects of a mutation within α helix3 and specific mutations within switch 1 and 2 differed among the GAPs. The largest differences were observed with a change of isoleucine 46 to aspartate ([I46D]Arf1), which reduced ASAP1-induced catalysis by ∼10,000-fold but had a 3-fold effect on AGAP1. The reduction was due to an isolated effect on the catalytic rate, kcat. In vivo [I46D]Arf1 had no detectable effect on the Golgi apparatus but, instead, functioned as a constitutively active mutant in the cell periphery, affecting the localization of ASAP1 and paxillin. Based on our results, we conclude that the contribution of specific residues within switch 1 of Arf to binding and achieving a transition state toward GTP hydrolysis differs among Arf GAPs

(m-Phenyl­enedimethyl­ene)diammonium p-nitro­phenyl­phosphate perchlorate

The title compound, C8H14N2 2+·C12H8N2O8P−·ClO4 −, was formed by the reaction of α,α-bis-m-xylenediamine and sodium bis-p-nitro­phenyl­phosphate in the presence of Zn(ClO4)·6H2O in methanol solution. The two amine groups of the m-xylenediammonium ion are each protonated and each hydrogen-bonded to two O atoms of the phosphate anion, which acts as a 1,3-bridge. The ammonium groups are arranged matched face to face and each pair is doubly bridged by two perchlorate ions through hydrogen bonding. In addition, there are also weak C—H⋯O inter­actions. Both the N—H⋯O and C—H⋯O inter­actions are contained in a channel down the a axis. The perchlorate oxygen atoms are disordered over two positions with site occupancy factors of ca 0.7 and 0.3

Three-dimensional structure of the human transglutaminase 3 enzyme: binding of calcium ions changes structure for activation

Transglutaminase (TGase) enzymes catalyze the formation of covalent cross-links between protein-bound glutamines and lysines in a calcium-dependent manner, but the role of Ca(2+) ions remains unclear. The TGase 3 isoform is widely expressed and is important for epithelial barrier formation. It is a zymogen, requiring proteolysis for activity. We have solved the three-dimensional structures of the zymogen and the activated forms at 2.2 and 2.1 Å resolution, respectively, and examined the role of Ca(2+) ions. The zymogen binds one ion tightly that cannot be exchanged. Upon proteolysis, the enzyme exothermally acquires two more Ca(2+) ions that activate the enzyme, are exchangeable and are functionally replaceable by other lanthanide trivalent cations. Binding of a Ca(2+) ion at one of these sites opens a channel which exposes the key Trp236 and Trp327 residues that control substrate access to the active site. Together, these biochemical and structural data reveal for the first time in a TGase enzyme that Ca(2+) ions induce structural changes which at least in part dictate activity and, moreover, may confer substrate specificity