GDP Binding Site Research Articles

New insights into P2X7 receptor regulation: Ca2+-calmodulin and GDP bind to the soluble P2X7 ballast domain

P2X7 receptors are nonselective cation channels that are activated by extracellular ATP and play important roles in inflammation. They differ from other P2X family members by a large intracellular C-terminus that mediates diverse signaling processes that are little understood. A recent cryo-EM study revealed that the C-terminus of the P2X7 receptor forms a unique cytoplasmic ballast domain that possesses a GDP-binding site as well as a dinuclear Zn2+ site. However, the molecular basis for the regulatory function of the ballast domain as well as the interplay between the various ligands remain unclear. Here, we successfully expressed a soluble trimeric P2X7 ballast domain (P2X7BD) and characterized its ligand binding properties using a biophysical approach. We identified calmodulin (CaM)-binding regions within the ballast domain and found that binding of Ca2+-CaM and GDP to P2X7BD have opposite effects on its stability. Small-angle X-ray scattering experiments indicate that Ca2+-CaM binding disrupts the trimeric state of P2X7BD. Our results provide a possible framework for the intracellular regulation of the P2X7 receptor.

Chemical IN04 Inhibits the Kinase Domain not the ROC Domain of LRRK1: Results from Homology Modeling and Molecular Docking.

Bone loss is the most common reason for broken bones among the elderly. An ideal agent for the treatment of bone loss should have both osteoclast inhibitory and osteoblast stimulatory functions. Leucine-rich repeat kinase 1 (LRRK1) is a novel target for alternative antiresorptive drugs to treat osteoporosis and osteoporotic fractures. Recently a chemical IN04, Methyl 3-[({([5-(3,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl]-thio}-acetyl)-amino]-benzoate, has been identified as a potential LRRK1 inhibitor. The aim of this work is to investigate how the chemical IN04 interacts with LRRK1 and inhibits its activity. A structural model of the LRRK1 kinase domain was constructed with SWISS-MODEL. The human protein kinase ROCO4 (PDB ID: 4YZN) was chosen as the template based on sequence homology, structural and phylogenetic analysis. In addition, a homology model of the LRRK1 ROC domain was also prepared based on the LRRK2 ROC domain structure (PDB ID: 2ZEJ). The interactions of IN04 with the active sites in the LRRK1 kinase domain and ROC domain were investigated by SwissDock. IN04 was docked into the active site of the LRRK1 kinase domain with similar interactions as ATP comparable to the ligand bound to homologous kinases. Many rational binding modes of IN04 to LRRK1 kinase domain were investigated and the most likely binding pose containing multiple hydrogen bonds and a salt bridge was discovered. However, IN04 cannot fit into the GDP-binding site of the ROC domain. Chemical IN04 inhibits LRRK1 by binding to the active site of the kinase domain but not the ROC domain.

Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to G\u03b1i1

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα.

Large-scale conformational rearrangement of the α5-helix of Gα subunits in complex with the guanine nucleotide exchange factor Ric8A

Resistance to inhibitors of cholinesterase 8A (Ric8A) protein is an important G protein-coupled receptor (GPCR)-independent regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor (GEF) and a chaperone. Insights into the complex between Ric8A and Gα hold the key to understanding the mechanisms underlying noncanonical activation of G-protein signaling as well as the folding of nascent Gα proteins. Here, we examined the structure of the complex of Ric8A with minimized Gαi (miniGαi) in solution by small-angle X-ray scattering (SAXS) and exploited the scattering profile in modeling of the Ric8A/miniGαi complex by steered molecular dynamics (SMD) simulations. A small set of models of the complex featured minimal clash scores, excellent agreement with the experimental SAXS data, and a large-scale rearrangement of the signal-transducing α5-helix of Gα away from its β-sheet core. The resulting interface involved the Gα α5-helix bound to the concave surface of Ric8A and the Gα β-sheet that wraps around the C-terminal part of the Ric8A armadillo domain, leading to a severe disruption of the GDP-binding site. Further modeling of the flexible C-terminal tail of Ric8A indicated that it interacts with the effector surface of Gα. This smaller interface may enable the Ric8A-bound Gα to interact with GTP. The two-interface interaction with Gα described here distinguishes Ric8A from GPCRs and non-GPCR regulators of G-protein signaling.

Structural and functional studies of phosphoenolpyruvate carboxykinase from Mycobacterium tuberculosis.

Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.

Correlation of transducin photoaffinity labeling with the specific formation of intermolecular disulfide linkages in its α-subunit.

Transducin (T) is a heterotrimer of Tα, Tβ, and Tγ subunits. In the presence of light-activated rhodopsin, 8-azidoguanosine triphosphate (8-N3GTP) was covalently incorporated into T in a UV-light photodependent manner, with a low stoichiometry of 0.02 mol of 8-N3GTP per mol of T. Although Tα was preferentially labeled by 8-N3GTP, Tβ and Tγ were also modified. Photolabeling of T was specifically inhibited by GDP and GTP, but not by β,γ-imido-guanosine 5'-triphosphate (GMP-PNP), indicating that 8-N3GTP was modifying the GDP binding site of the holoenzyme. This was consistent with the observation that the photoaffinity probe was completely hydrolyzed to 8-N3GDP by T activated by illuminated rhodopsin. The formation of intermolecular disulfide associations in T was also determined because photolabeling of T was performed under non-reducing conditions. We established that Cys-347 of Tα was the major residue involved in the formation of disulfide-linked T oligomers. Other cysteines of Tα, such as Cys-321, also participated in the formation of disulfide bonds, revealing a complex pattern of intermolecular disulfide cross-links that led to the polymerization of T. The spontaneous generation of these cystines in Tα inhibited the light-dependent GTPase and GMP-PNP binding activities of T. A model was constructed illustrating that when two heterotrimers dimerize through the formation of disulfide bridges between the Cys-347 of their Tα subunits, the guanine ring of the 8-N3GDP bound to one T molecule might approach to the Tβγ-complex of the other heterotrimer. This model provides an explanation for the additional photolabeling of Tβ and Tγ by 8-N3GTP.

Structural Coupling between the Rho-Insert Domain of Cdc42 and the Geranylgeranyl Binding Site of RhoGDI

The small GTPase proteins are components of the intracellular signaling system, alternating between active (membrane-bound and GTP-loaded) and inactive (GDP-loaded and cytosolic) states. In the inactive state, the proteins are soluble in the cytoplasm. To compensate for the energetic penalty of extraction of the hydrophobic moiety from the membrane phase, the inactive state is stabilized via formation of a complex with the RhoGDI proteins that provide a hydrophobic pocket for the binding of the hydrophobic moieties. The signals delivered by the Rho subfamily involve a specific, short, highly exposed α-helix (Rho-insert), located close to the GDP binding site. Upon simulating the complex in solution, we observed that the Rho-insert domain of Cdc42 can assume two basic orientations. One is the canonical one, as detected in both crystals and NMR spectra of concentrated protein solutions. The second orientation appears only in the RhoGDI-Cdc42 complex where the GER moiety of Cdc42 is properly inserted into the specific binding site of RhoGDI. Any impairment of the GER-RhoGDI interactions, such as insertion of specific mutations in the hydrophobic binding site, abolished the coupling between the proteins and the Rho-insert domain, preserving its canonical orientation as in the crystalline structure. The noncanonical conformation of the Rho-insert domain is not a simulation artifact, as it appears in crystals of plant Rho proteins (ROP4, ROP5, and ROP7). In accord with the notion that the Rho-insert domain participates in downstream signaling, we propose that the deformation of the Rho-insert is part of the signal transmissions.

Binding of GTPγ[35S] is regulated by GDP and receptor activation. Studies with the nociceptin/orphanin FQ receptor

Background and purpose: We have examined the effects of ligand efficacy and receptor density on the binding of guanosine 5′‐[γ‐thio]triphosphate (GTPγS) and GDP to the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP)‐coupled G‐proteins.Experimental approach: In GTPγ[35S] binding experiments, using stable (CHOhNOP) and inducible (CHOINDhNOP) recombinant human and rat NOP we have measured: (i) ligand‐specific GDP requirements; (ii) the effects of receptor density on guanine nucleotide affinity/capacity; and (iii) the effect of ligand efficacy on GTPγS association kinetics.Key results: GTPγS competition curves were shallow and modelled by high‐ and low‐affinity components that were relatively consistent between cell types and tissue preparations. In the presence of 1 µM N/OFQ a high‐affinity GDP binding site was also present, but the fraction of total binding was reduced. In an efficacy‐dependent manner, the partial agonists [F/G]N/OFQ(1‐13)NH2 ([Phe1ψ(CH2‐NH)Gly2]‐nociceptin(1‐13)NH2) and naloxone benzoylhydrazone both reduced the fraction of high‐affinity sites for GDP (relative to basal). While the pIC50 for high‐affinity GDP binding site did not decrease in the presence of 1 µM N/OFQ, N/OFQ produced a significant reduction in pIC50 for the low‐affinity site. Agonist‐mediated decrease in affinity for GDP binding was efficacy‐dependent. GDP displayed three affinities: high, conserved in the presence and absence of ligand; intermediate, present as a low fraction under basal conditions; low (efficacy‐dependent), present during receptor activation representing the majority of binding.Conclusions and implications: The affinity of GTPγ[35S] was regulated by GDP and receptor activation caused increased binding of GTPγ[35S] through a reduction in GDP affinity.

Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA

GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.

Role of the individual domains of translation termination factor eRF1 in GTP binding to eRF3

Eukaryotic translational termination is triggered by polypeptide release factors eRF1, eRF3, and one of the three stop codons at the ribosomal A-site. Isothermal titration calorimetry shows that (i) the separated MC, M, and C domains of human eRF1 bind to eRF3; (ii) GTP binding to eRF3 requires complex formation with either the MC or M + C domains; (iii) the M domain interacts with the N and C domains; (iv) the MC domain and Mg2+ induce GTPase activity of eRF3 in the ribosome. We suggest that GDP binding site of eRF3 acquires an ability to bind gamma-phosphate of GTP if altered by cooperative action of the M and C domains of eRF1. Thus, the stop-codon decoding is associated with the N domain of eRF1 while the GTPase activity of eRF3 is controlled by the MC domain of eRF1 demonstrating a substantial structural uncoupling of these two activities though functionally they are interrelated.

Molecular Basis of Guanine Nucleotide Dissociation Inhibitor Activity of Human Neuroglobin by Chemical Cross-linking and Mass Spectrometry

Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein α-subunit (Gα i). Here, to elucidate the molecular mechanism underlying the GDI activity of Ngb, we used an glutathione- S-transferase pull-down assay to confirm that Ngb competes with G-protein βγ-subunits (Gβγ) for binding to Gα i, and identified the Gα i-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo- N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-Gα i complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Gα i peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (Gα i), and between Glu53 (Ngb) and Ser44 (Gα i). Because Ser206 of Gα i is located in the region that contacts Gβγ, binding of Ngb could facilitate the release of Gβγ from Gα i. Binding of Ngb to Gα i would also inhibit the exchange of GDP for GTP, because Ser44 (Gα i) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (Gα i), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Gα i, enabling us to discuss the functional significance of this binding on the GDI activity of Ngb.

Biochemical and Structural Characterization of the Gem GTPase

RGK proteins, encompassing Rad, Gem, Rem1, and Rem2, constitute an intriguing branch of the Ras superfamily; their expression is regulated at the transcription level, they exhibit atypical nucleotide binding motifs, and they carry both large N- and C-terminal extensions. Biochemical and structural studies are required to better understand how such proteins function. Here, we report the first structure for a RGK protein: the crystal structure of a truncated form of the human Gem protein (G domain plus the first part of the C-terminal extension) in complex with Mg.GDP at 2.1 A resolution. It reveals that the G-domain fold and Mg.GDP binding site of Gem are similar to those found for other Ras family GTPases. The first part of the C-terminal extension adopts an alpha-helical conformation that extends along the alpha5 helix and interacts with the tip of the interswitch. Biochemical studies show that the affinities of Gem for GDP and GTP are considerably lower (micromolar range) compared with H-Ras, independent of the presence or absence of N- and C-terminal extensions, whereas its GTPase activity is higher than that of H-Ras and regulated by both extensions. We show how the bulky DXWEX motif, characteristic of the switch II of RGK proteins, affects the conformation of switch I and the phosphate-binding site. Altogether, our data reveal that Gem is a bona fide GTPase that exhibits striking structural and biochemical features that should impact its regulation and cellular activities.

Integrating Three Views of Arf1 Activation Dynamics

The proteins Arno and Gea2 of the Sec7 family can promote GDP–GTP exchange on Arf1, a small GTP-binding protein, which coordinates coated vesicle formation for protein transport within the cell. Crystal structures of the essential Sec7 domain (Sec7d) of Gea2 in the free and Arf1-bound forms suggest that conformational dynamics of the Sec7d as well as those of the G-protein play a role in nucleotide exchange. Starting from a set of complementary crystal structures, we compared the collective movements of unbound Gea2 and Arno Sec7 domains, Arf1-GDP, and the Arf1–Gea2(Sec7d) nucleotide-free complex using normal modes analyses. In all unbound Sec7d analyses, significant low-energy movements were found to lead to closure of the hydrophobic groove towards the form seen in the Arf1–Gea2(Sec7d) complex, suggesting that groove closure is a general feature of the Sec7 family. Low-energy movements in Arf1-GDP implicate critical switch 1 and 2 residues which are coupled to modifications in the myristoylated N-terminal-helix binding site at the other end of the “interswitch” beta hairpin. It is suggested that Sec7d groove closure upon docking of the two molecules may permit extraction of switch 1 from Arf1-GDP and prepare the complex for movement of the interswitch, which is central to the membrane-linked exchange activity. Large-scale collective movements in the Arf1–Sec7d complex appear to participate in the insertion of the Sec7d Glu finger into the GDP binding site to promote actual nucleotide release.

Domain Arrangement of Der, a Switch Protein Containing Two GTPase Domains

The EngA subfamily of essential bacterial GTPases has a unique domain structure consisting of two adjacent GTPase domains (GD1 and GD2) and a C-terminal domain. The structure of Thermotoga maritima Der bound to GDP determined at 1.9 Å resolution reveals a novel domain arrangement in which the GTPase domains pack at either side of the C-terminal domain. Unexpectedly, the C-terminal domain resembles a KH domain, missing the distinctive RNA recognition elements. Conserved motifs of the nucleotide binding site of GD1 are integral parts of the GD1-KH domain interface, suggesting the interactions between these two domains are directly influenced by the GTP/GDP cycling of the protein. In contrast, the GD2-KH domain interface is distal to the GDP binding site of GD2.

The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with GDP reveals novel features in nucleotide binding and exchange

The crystal structure of elongation factor 1alpha from the archaeon Sulfolobus solfataricus in complex with GDP (SsEF-1alpha.GDP) at 1.8 A resolution is reported. As already known for the eubacterial elongation factor Tu, the SsEF-1alpha.GDP structure consists of three different structural domains. Surprisingly, the analysis of the GDP-binding site reveals that the nucleotide- protein interactions are not mediated by Mg(2+). Furthermore, the residues that usually co-ordinate Mg(2+) through water molecules in the GTP-binding proteins, though conserved in SsEF-1alpha, are located quite far from the binding site. [(3)H]GDP binding experiments confirm that Mg(2+) has only a marginal effect on the nucleotide exchange reaction of SsEF-1alpha, although essential to GTPase activity elicited by SsEF-1alpha. Finally, structural comparisons of SsEF- 1alpha.GDP with yeast EF-1alpha in complex with the nucleotide exchange factor EF-1beta shows that a dramatic rearrangement of the overall structure of EF-1alpha occurs during the nucleotide exchange.

High resolution crystal structure of bovine mitochondrial EF-tu in complex with GDP

The crystal structure of bovine mitochondrial elongation factor Tu (EF-Tu) in complex with GDP has been determined at a resolution of 1.94 Å. The structure is similar to that of EF-Tu:GDP from Escherichia coli and Thermus aquaticus, but the orientation of the GDP-binding domain 1 is changed relative to domains 2 and 3. Sixteen conserved water molecules common to EF-Tu and other G-proteins in the GDP-binding site are described. These water molecules create a network linking separated parts of the binding pocket. Mitochondrial EF-Tu binds nucleotides less tightly than prokaryotic EF-Tu possibly due to an increased mobility in regions close to the GDP-binding site. The C-terminal extension of mitochondrial EF-Tu has structural similarities with DNA recognising zinc fingers suggesting that the extension may be involved in recognition of RNA.

Conformational Changes at The Carboxyl Terminus of Gα Occur during G Protein Activation

To understand the dynamics of conformational changes during G protein activation, surface exposed cysteine residues on Galpha were fluorescently labeled. Limited trypsinolysis and mutational analysis of recombinant Galphat/Galphai1 determined that two cysteines are the major fluorescent labeling sites, Cys210, located in the switch II region, and Cys347 at the C terminus. Mutants with serines replacing Cys210 (Chi6a) and Cys347 (Chi6b) were single fluorescently labeled with lucifer yellow (LY), while a double mutant (Chi6ab) was no longer labeled. When Chi6b was labeled with LY on Cys210, AlF4- caused a 220% increase in LY fluorescence, indicating that the fluorescent group at Cys210 is a reporter of conformational change in the switch II region. Chi6a labeled at Cys347 also showed an AlF4--dependent increase in LY fluorescence (91%), indicating that Galpha activation leads to a conformational change at the COOH terminus. Preactivation of the protein with AlF4- before labeling led to a decreased incorporation of LY into Cys347 suggesting that Galpha activation buries Cys347. This COOH-terminal conformational change may provide the structural basis for communication between the GDP-binding site on Galpha and activated receptors, and may contribute to dissociation of activated Galpha subunit from activated receptor.

The A326S Mutant of Giα1 as an Approximation of the Receptor-bound State

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their alpha subunits. In search of an approximation of the receptor-alpha subunit complex, we have considered the properties of A326S Gialpha1, a mutation discovered originally in Gsalpha (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the alphai1beta1gamma2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Gialpha1 for GTPgammaS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the alpha subunit. The mutation also uncouples two activities of betagamma: stabilization of the GDP-bound alpha subunit (which is retained) and retardation of GDP dissociation from the heterotrimer (which is lost). For wild-type and mutant Gialpha1, beta gamma prevents irreversible inactivation of the alpha subunit at 30 degreesC. However, the mutation accelerates irreversible inactivation of alpha at 37 degreesC despite the presence of beta gamma. Structurally, the mutation weakens affinity for GTPgammaS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S Gialpha1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S Gialpha1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a Galpha subunit in the GDP-bound state.

Sulfonates are low-affinity ligands for the GDP-binding site of brown-fat mitochondria

In order to study the function of the brown-fat specific uncoupling protein thermogenin (UCP), the effect of certain sulfonates on [ 3H]GDP binding to the GDP-binding site of brown adipose tissue mitochondria was studied. The affinity of [ 3H]GDP for the site was 1.3 μM in the normal sucrose medium, but the apparent K D was increased to ≈ 20 μM in 100 mM hexanesulfonate medium. This increase in apparent K D was found to be due to a competitive binding of hexanesulfonate to the GDP-binding site; the affinity of hexanesulfonate was only 13 mM but this was sufficient to affect the apparent affinity of GDP under experimental conditions. Also in KCl-medium, the affinity of GDP was high (≈ 3 μM), but both in a benzenesulfonate medium and in a para-aminobenzenesulfonate (sulfanilate) medium, the apparent affinity was lower (≈ 12 μM); as benzenesulfonate is well transported by thermogenin but sulfanilate is not, the reduction in affinity was unrelated to transport. In agreement with earlier data (Jezek, P. and Garlid, K.D. (1990) J. Biol. Chem. 265, 19303–19311), the potency of GDP to inhibit transport was dependent on the species transported; the fact that GDP potency was lower for benzenesulfonate transport (EC 50 = 324 μM) than for Cl − transport (EC 50 = 32 μM) could adequately be explained by the competitive interaction of benzenesulfonate with the GDP-binding site, but this effect could only partly explain the even lower potency of GDP to inhibit hexanesulfonate transport (EC 50 = 4074 μM). It was concluded that these types of substrate for thermogenin-mediated transport may directly interact with the GDP-binding site, but that this effect could only partly explain in the dependence of GDP potency on substrate species.

SPROUT: recent developments in the de novo design of molecules.

SPROUT is a computer program for constrained structure generation. It is designed to generate molecules for a range of applications in molecular recognition. The program uses a number of approximations that enable a wide variety of diverse structures to be generated. Practical use of the program is demonstrated in two examples. The first demonstrates the ability of the program to generate candidate inhibitors for a receptor site of known 3D structure, specifically the GDP binding site of p21. In the second example, structures are generated to fit a pharmacophore hypothesis that models morphine agonists.