Guanine nucleotide-binding (G) proteins, which cycle between a GDP- and a GTP-bound conformation, are conventionally regulated by GTPase-activating proteins (GAPs) and guanine nucleotide-exchange factors (GEFs), and function by interacting with effector proteins in the GTP-bound 'on' state. Here we present another class of G proteins that are regulated by homodimerization, which we would categorize as G proteins activated by nucleotide-dependent dimerization (GADs). This class includes proteins such as signal recognition particle (SRP), dynamin, septins and the newly discovered Roco protein Leu-rich repeat kinase 2 (LRRK2). We propose that the juxtaposition of the G domains of two monomers across the GTP-binding sites activates the biological function of these proteins and the GTPase reaction.

The retinitis pigmentosa 2 (RP2) gene is responsible for a particular variant of X chromosome-linked eye disease. Previously, RP2 was shown to bind the GTP form of the small G protein Arf-like 3 (Arl3), thus qualifying as an effector. Here we present the Arl3-GppNHp-RP2 complex structure, which shows features resembling complexes with GTPase-activating proteins (GAPs). Biochemical analysis showing a 90,000-fold stimulation of the GTPase reaction together with the structure of an Arl3-GDP-AlF4--RP2 transition state complex showed that RP2 is an efficient GAP for Arl3, with structural features similar to other GAPs. Furthermore, the effect of mutations in patients with retinitis pigmentosa correlated with their effect on catalysis, in particular the mutation of the arginine finger of RP2. The cognate G protein-GAP pair is conserved in yeast as Cin4-Cin2, and the ability of RP2 to act as a GAP can be correlated with its ability to complement a CIN2-deletion phenotype.

HypB is a prokaryotic metal-binding guanine nucleotidebinding protein that is essential for nickel incorporation into hydrogenases. Here we solved the x-ray structure of HypB from Methanocaldococcus jannaschii. It shows that the G-domain has a different topology than the Ras-like proteins and belongs to the SIMIBI (after Signal Recognition Particle, MinD and BioD) class of NTP-binding proteins. We show that HypB undergoes nucleotide-dependent dimerization, which is apparently a common feature of SIMIBI class G-proteins. The nucleotides are located in the dimer interface and are contacted by both subunits. The active site features residues from both subunits arguing that hydrolysis also requires dimerization. Two metal-binding sites are found, one of which is dependent on the state of bound nucleotide. A totally conserved ENV/IGNLV/ICP motif in switch II relays the nucleotide binding with the metal ionbinding site. The homology with NifH, the Fe protein subunit of nitrogenase, suggests a mechanistic model for the switch-dependent incorporation of a metal ion into hydrogenases.Maturation of nickel-containing enzymes, like carbon monoxide dehydrogenase, urease, or hydrogenase, requires participation of numerous accessory proteins. Sequencing of several bacterial genomes combined with genetic and biochemical analysis revealed a set of such proteins for each metalloenzyme (1, 2). Although the basic functions of nickel storage, GTP hydrolysis-dependent nickel mobilization, and nickel insertion have been demonstrated, the specific steps performed by these proteins in the assembly of the active site are not completely understood (3). In the case of hydrogenases, the generation of active enzyme that also involves nickel incorporation is achieved by the activities of six proteins designated HypA-F (4). In most organisms the corresponding genes are encoded in a single operon. However, genes for additional maturation proteins, also including a maturation endopeptidase, are normally organized in an operon that also houses the hydrogenase structural genes (5). Two of these auxiliary proteins, namely HypA and HypB, are required for the nickel insertion reaction as judged by the fact that the deficiency of hypA and hypB mutants in hydrogenase maturation could be complemented chemically by the addition of nickel to the medium in high concentrations or by the addition of the metal to maturation assays employing crude extracts (6 -8). The same mutations also reduce efficiency of urease maturation in Helicobacter pylori showing the more general importance of the two proteins (9).Genetically, HypA and HypB are the key factors for enzymatic activity because mutations in one of these results in hydrogenase deficiency (7,8). The same mutations also reduce maturation efficiency of urease showing the more general importance of the two proteins (9). HypA is capable of binding two nickel ions per dimer with micromolar affinity (10) and two zinc ions with much higher affinity, which are supposed to be necessary for structural integrity of t...

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all- trans retinal in photoexcited bR to a highly twisted 13- cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins.