Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.

Methionine-rich motifs have an important role in copper trafficking factors, including the CusF protein. Here we show that CusF uses a new metal recognition site wherein Cu(I) is tetragonally displaced from a Met 2 His ligand plane toward a conserved tryptophan. Spectroscopic studies demonstrate that both thioether ligation and strong cation-π interactions with tryptophan stabilize metal binding. This novel active site chemistry affords mechanisms for control of adventitious metal redox and substitution chemistry.In recent years, metal-specific gene regulatory and cation-trafficking proteins have been isolated and demonstrate metal binding motifs with unprecedented coordination chemistry tailored to their function 1 . For example, the CXXC sequence, found in cytosolic copper chaperones and trafficking proteins, provides for facile Cu(I) transfer via low-coordinationnumber anionic intermediates 1,2 . Extracellular or periplasmic copper trafficking domains, however, function in environments that are more oxidizing than the cytosol and frequently have less well understood methionine-rich sequences 3-8 . The cus operon encodes a bacterial copper homeostasis system with several methionine-motif proteins 5,9,10 , including the periplasmic protein CusF, which is thought to serve as copper chaperone or regulator 5,6 . CusF binds Cu(I) in vitro 11 , and a methionine-rich Cu(I) site was proposed 6 based on an apo-CusF structure and NMR chemical shift data. Here we show that metal recognition in CusF involves a strong interaction between a cationic Cu(I)-thioether/imidazole center and the aromatic ring of tryptophan. To our knowledge, such cation-π interactions have not been reported for transition metal receptors or metalloenzyme active sites.Correspondence should be addressed to T.V.O. (t-ohalloran@northwestern.edu). 6 These authors contributed equally to this work.Published online at http://www.nature.com/naturechemicalbiology Reprints and permissions information is available online at

Potassium channels catalyze the selective transfer of potassium across the cell membrane and are essential for setting the resting potential in cells, controlling heart rate and modulating the firing pattern in neurons. Tetraethylammonium (TEA) blocks ion conduction through potassium channels in a voltage-dependent manner from both sides of the membrane. Here we show the structural basis of TEA blockade by cocrystallizing the prokaryotic potassium channel KcsA with two selective TEA analogs. TEA binding at both sites alters ion occupancy in the selectivity filter; these findings underlie the mutual destabilization and voltage-dependence of TEA blockade. We propose that TEA blocks potassium channels by acting as a potassium analog at the dehydration transition step during permeation.

beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and the cephalosporins. In an effort to combat these enzymes, a combination of stereoselective organic synthesis, enzymology, microbiology, and X-ray crystallography was used to design and evaluate new carboxyphenyl-glycylboronic acid transition-state analogue inhibitors of the class C beta-lactamase AmpC. The new compounds improve inhibition by over 2 orders of magnitude compared to analogous glycylboronic acids, with K(i) values as low as 1 nM. On the basis of the differential binding of different analogues, the introduced carboxylate alone contributes about 2.1 kcal/mol in affinity. This carboxylate corresponds to the ubiquitous C3(4)' carboxylate of beta-lactams, and this energy represents the first thermodynamic measurement of the importance of this group in molecular recognition by class C beta-lactamases. The structures of AmpC in complex with two of these inhibitors were determined by X-ray crystallography at 1.72 and 1.83 A resolution. These structures suggest a structural basis for the high affinity of the new compounds and provide templates for further design. The highest affinity inhibitor was 5 orders of magnitude more selective for AmpC than for characteristic serine proteases, such as chymotrypsin. This inhibitor reversed the resistance of clinical pathogens to the third generation cephalosporin ceftazidime; it may serve as a lead compound for drug discovery to combat bacterial resistance to beta-lactam antibiotics.

Hypoxanthine phosphoribosyltransferase (HPRT) salvages 6-oxopurine bases in the nucleotide metabolic pathway. The 1.8 A crystal structure of an asymmetric dimer of the HPRT from the protozoan parasite Trypanosoma cruzi was determined in a ternary complex with the primary substrate phosphoribosylpyrophosphate (PRPP) and an analogue of the substrate hypoxanthine, revealing both open and closed active site conformations. The ligands are positioned for in-line nucleophilic attack at the PRPP ribose C1' by two metal ions which straddle the pyrophosphate leaving group. The structure provides the first evidence for the involvement of two metal ions in the HPRT-catalyzed reaction, and structural details further suggest the mechanism may proceed via SN2-type chemistry. The closed conformation reveals the structural roles for invariant flexible loop residues Ser103 and Tyr104 and supports a role for the loop in the liberation of pyrophosphate. The pre-transition state structure is valuable for understanding the enzyme mechanism, as well as providing a foundation for antiparasite drug design efforts against T. cruzi, which causes Chagas' disease in humans. Additionally, the structure illuminates the molecular basis of three inherited mutations in the human HPRT leading to Lesch-Nyhan syndrome (D193N) or gout (S103R or S109L), as the homologous residues in the trypanosomal enzyme contribute to the previously unrecognized Mg2+ ion binding site and to the formation of the closed flexible loop, respectively.