Inhibition of Subsets of G Protein-coupled Receptors by Empty Mutants of G Protein α Subunits in Go, G11, and G16

We previously reported that the xanthine nucleotide binding Goα mutant, GoαX, inhibited the activation of Gi-coupled receptors. We constructed similar mutations in G11α and G16α and characterized their nucleotide binding and receptor interaction. First, we found that G11αX and G16αX expressed in COS-7 cells bound xanthine 5'-O-(thiotriphosphate) instead of guanosine 5'-O-(thiotriphosphate). Second, we found that G11αX and G16αX interacted with βγ subunits in the presence of xanthine diphosphate. These experiments demonstrated that G11aαX and G16αX were xanthine nucleotide-binding proteins, similar to GoαX. Third, in COS-7 cells, both G11αX and G16αX inhibited the activation of Gq-coupled receptors, whereas only G16αX inhibited the activation of Gi-coupled receptors. Therefore, when in the nucleotide-free state, empty G11αX and G16αX appeared to retain the same receptor binding specificity as their wild-type counterparts. Finally, we found that GoαX, G11αX, and G16αX all inhibited the endogenous thrombin receptors and lysophosphatidic acid receptors in NIH3T3 cells, whereas G11αX and G16αX, but not GoαX, inhibited the activation of transfected m1 muscarinic receptor in these cells. We conclude that these empty G protein mutants of Goα, G11α, and G16α can act as dominant negative inhibitors against specific subsets of G protein-coupled receptors

The properties of Msh2-Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair.

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2-Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2-Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2-Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide-binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2-Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo The mutant Msh2-Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1-Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1-Pms1-independent and Mlh1-Pms1-dependent MMR reactions in vitro and in activation of the Mlh1-Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2-Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR

Gz, a guanine nucleotide-binding protein with unique biochemical properties

Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26- kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP- ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min^-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min^-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family

CFTR Gating II: Effects of Nucleotide Binding on the Stability of Open States

Previously, we demonstrated that ADP inhibits cystic fibrosis transmembrane conductance regulator (CFTR) opening by competing with ATP for a binding site presumably in the COOH-terminal nucleotide binding domain (NBD2). We also found that the open time of the channel is shortened in the presence of ADP. To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Single-channel kinetic analysis of ΔR/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. ΔR/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of ∼100 s, estimated from current relaxation upon nucleotide removal. ADP induces a shorter locked-open state, suggesting that binding of ADP at a second site decreases the locked-open time. To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Two locked-open time constants can be discerned and the relative distribution of each component is altered by changing [ATP] so that increasing [ATP] shifts the relative distribution to the longer locked-open state. Single-channel kinetic analysis for ΔR/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. These results support the idea that occupancy of a second ATP binding site stabilizes the locked-open state. This binding site likely resides in the NH(2)-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. These results suggest that the binding energy of nucleotide at NBD1 contributes to the overall energetics of the open channel conformation

Structural basis of the allosteric trigger of the Hsp70 chaperone proteins.

This work solves a decades-old dilemma that stood in the way of understanding the allosteric mechanism of Hsp70 (heat shock 70 kDa) chaperone proteins. Hsp70s are central to protein folding, refolding, and trafficking in organisms ranging from Archae to Homo Sapiens, both at normal and at stressed cellular conditions. Hsp70s are comprised of two main domains: a 44 kDa N-terminal nucleotide-binding domain (NBD), and a 25 kDa substrate-binding domain (SBD) that harbors the substrate binding site. The nucleotide binding site in the NBD and the substrate binding site in the SBD are allosterically linked: ADP binding promotes substrate binding, while ATP binding promotes substrate release. It has long been a goal of structural biology to characterize the nature of the allosteric coupling in these proteins. However, even the most sophisticated X-ray crystallography studies of the isolated NBD could show no difference in overall conformation between the ATP and ADP state. Hence the dilemma: how is the state of the nucleotide communicated between NBD and SBD? The solution of the dilemma is especially interesting in light of the fact that Hsp70s are ancient proteins, and amongst the first allosteric proteins in nature.Here we report a solution NMR study of the NBD of the Hsp70 from Thermus thermophilus, in the APO, ADP and AMP-PNP states, where the latter is a non-hydrolysable ATP analogue. Using the modern NMR methods of residual dipolar coupling analysis, we discovered that the nucleotide binding cleft opens up by as much as 20 degrees between the AMP-PNP (closed) and ADP (open) state. We also discover that a surface cleft, hypothesized to be essential for the allosteric coupling between NBD and SBD, echoes these changes. Hence, the nature of the allosteric trigger and coupling for Hsp70 chaperones is revealed here for the first time, solving the dilemma

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.

Nucleotide-Binding Sites of the Heterodimeric LmrCD ABC-Multidrug Transporter of Lactococcus lactis Are Asymmetric

LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[α-32P]ATP, which, at 4 °C, occurs predominantly at LmrC, while aluminiumfluoride (AlFx)-induced trapping of the hydrolyzed nucleotide at 30 °C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs.

Identifizierung und Charakterisierung eines neuen Bindeproteins für zyklische Nukleotide

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate are important intracellular messengers. Binding of cyclic nucleotides controls the activity of protein kinases, ion channels and guanine-nucleotide-exchange factors in many cells. The SCNBP (soluble cyclic nucleotide-binding protein) is a novel uncharacterized protein predicted to comprise a cyclic nucleotide-binding domain. This protein belongs to neither of the known families of effector proteins for cyclic nucleotides. Within 17 distinct species - from marine invertebrates to humans - genes orthologous to the mouse SCNBP are present. Hence, the SCNBP could belong to a novel class of effector proteins for cyclic nucleotides. Northern blot experiments with mouse tissue indicate that the mRNA of SCNBP is expressed predominantly in the testis and by means of in situ hybridization it was specifically detected in spermatocytes. In the present study, SCNBP expression has been analyzed in mouse testis utilizing specific antibodies. I could provide evidence that two distinct SCNBP variants are present in mouse testis. To approach the physiological function of SCNBP, I identified by immunoprecipitation and mass spectrometry proteins in mouse testis that potentially interact with SCNBP. For a comprehensive biochemical study, SCNBP was heterologously expressed in Chinese hamster ovary (CHO) cells. Following fermentation of these cells in a stirred tank bioreactor I purified SCNBP by affinity chromatography

Photocontrol of the GTPase activity of the small G protein K-Ras by using an azobenzene derivative

AbstractThe small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Switch mechanisms utilizing conformational changes in nucleotide-binding motifs have been well studied at the molecular level. Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis and trans forms upon exposure to ultraviolet and visible light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the nucleotide-binding motif of Ras to regulate the GTPase activity by photoirradiation. We prepared four Ras mutants (G12C, Y32C, I36C, and Y64C) that have a single reactive cysteine residue in the nucleotide-binding motif. PAM was stoichiometrically incorporated into the cysteine residue of the mutants. The PAM-modified mutants exhibited reversible alterations in GTPase activity, nucleotide exchange rate, and interaction between guanine nucleotide exchange factor and Ras, accompanied by photoisomerization upon exposure to ultraviolet and visible light irradiation. The results suggest that incorporation of photochromic molecules into its nucleotide-binding motif enables photoreversible control of the function of the small G protein Ras