GPCR activation:protonation and membrane potential

GPCR proteins represent the largest family of signaling membrane proteins in eukaryotic cells.Their importance to basic cell biology,human diseases,and pharma-ceutical interventions is well established.Many crystal structures of GPCR proteins have been reported in both active and inactive conformations.These data indicate that agonist binding alone is not suffi cient to trigger the conformational change of GPCRs necessary for binding of downstream G-proteins,yet other essential factors re-main elusive.Based on analysis of available GPCR crystal structures,we identifi ed a potential conformational switch around the conserved Asp2.50,which consistently shows distinct conformations between inactive and active states.Combining the structural information with the current literature,we propose an energy-coupling mechanism,in which the interaction between a charge change of the GPCR protein and the membrane potential of the living cell plays a key role for GPCR activation.

Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit

Nascent polypeptide associated complex(NAC)and its two isolated subunits,αNAC and βNAC,play important roles in nascent peptide targeting.We determined a 1.9Åresolution crystal structure of the interaction core of NAC heterodimer and a 2.4Åresolution crystal structure ofαNAC NAC domain homodimer.These structures provide detailed information of NAC heterodimerization and αNAC homodimerization.We found that the NAC domains of αNAC and βNAC share very similar folding despite of their relative low identity of amino acid sequences.Furthermore,different electric charge distributions of the two subunits at the NAC interface provide an explanation to the observation that the heterodimer of NAC complex is more stable than the single subunit homodimer.In addition,we successfully built a βNAC NAC domain homodimer model based on homologous modeling,suggesting that NAC domain dimerization is a general property of the NAC family.These 3D structures allow further studies on structurefunction relationship of NAC.

Structural study of the Cdc25 domain from Ral-specific guanine-nucleotide exchange factor RalGPS1a

The guanine-nucleotide exchange factor(GEF)RalGPS1a activates small GTPase Ral proteins such as RalA and RalB by stimulating the exchange of Ral bound GDP to GTP,thus regulating various downstream cellular processes.RalGPS1a is composed of an Nterminal Cdc25-like catalytic domain,followed by a PXXP motif and a C-terminal pleckstrin homology(PH)domain.The Cdc25 domain of RalGPS1a,which shares about 30%sequence identity with other Cdc25-domain proteins,is thought to be directly engaged in binding and activating the substrate Ral protein.Here we report the crystal structure of the Cdc25 domain of RalGPS1a.The bowl shaped structure is homologous to the Cdc25 domains of SOS and RasGRF1.The most remarkable difference between these three Cdc25 domains lies in their active sites,referred to as the helical hairpin region.Consistent with previous enzymological studies,the helical hairpin of RalGPS1a adopts a conformation favorable for substrate binding.A modeled RalGPS1a-RalA complex structure reveals an extensive binding surface similar to that of the SOS-Ras complex.However,analysis of the electrostatic surface potential suggests an interaction mode between the RalGPS1a active site helical hairpin and the switch 1 region of substrate RalA distinct from that of the SOS-Ras complex.

Structure analysis of the extracellular domain reveals disulfide bond forming-protein properties of Mycobacterium tuberculosis Rv2969c

Disulfide bond-forming(Dsb)protein is a bacterial periplasmic protein that is essential for the correct folding and disulfide bond formation of secreted or cell wallassociated proteins.DsbA introduces disulfi de bonds into folding proteins,and is re-oxidized through interaction with its redox partner DsbB.Mycobacterium tuberculosis,a Gram-positive bacterium,expresses a DsbA-like protein(Rv2969c),an extracellular protein that has its Nterminus anchored in the cell membrane.Since Rv2969c is an essential gene,crucial for disulfi de bond formation,research of DsbA may provide a target of a new class of anti-bacterial drugs for treatment of M.tuberculosis infection.In the present work,the crystal structures of the extracellular region of Rv2969c(Mtb DsbA)were determined in both its reduced and oxidized states.The overall structure of Mtb DsbA can be divided into two domains:a classical thioredoxin-like domain with a typical CXXC active site,and anα-helical domain.It largely resembles its Escherichia coli homologue EcDsbA,however,it possesses a truncated binding groove;in addition,its active site is surrounded by an acidic,rather than hydrophobic surface.In our oxidoreductase activity assay,Mtb DsbA exhibited a different substrate specifi city when compared to EcDsbA.Moreover,structural analysis revealed a second disulfi de bond in Mtb DsbA,which is rare in the previously reported DsbA structures,and is assumed to contribute to the overall stability of Mtb DsbA.To investigate the disulphide formation pathway in M.tuberculosis,we modeled Mtb Vitamin K epoxide reductase(Mtb VKOR),a binding partner of Mtb DsbA,to Mtb DsbA.

Crystal structures and biochemical studies of human lysophosphatidic acid phosphatase type 6

Lysophosphatidic acid(LPA)is an important bioac-tive phospholipid involved in cell signaling through G-protein-coupled receptors pathways.It is also involved in balancing the lipid composition inside the cell,and modulates the function of lipid rafts as an intermediate in phospholipid metabolism.Because of its involvement in these important processes,LPA degradation needs to be regulated as precisely as its production.Lysophospha-tidic acid phosphatase type 6(ACP6)is an LPA-specifi c acid phosphatase that hydrolyzes LPA to monoacylglyc-erol(MAG)and phosphate.Here,we report three crystal structures of human ACP6 in complex with malonate,L-(+)-tartrate and tris,respectively.Our analyses revealed that ACP6 possesses a highly conserved Rossmann-fold-like body domain as well as a less conserved cap domain.The vast hydrophobic substrate-binding pocket,which is located between those two domains,is suitable for ac-commodating LPA,and its shape is different from that of other histidine acid phosphatases,a fact that is consistent with the observed difference in substrate preferences.Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis.The structure also indicates a water-supplying channel for substrate hydrolysis.Our structural data are consistent with the fact that the enzyme is active as a monomer.In combination with additional mutagenesis and enzyme activity studies,our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.