Thermodynamics and dynamics of histidine-binding protein, the water-soluble receptor of histidine permease. Implications for the transport of high and low affinity ligands.

The bacterial histidine permease is a model system for ABC transporters (traffic ATPases). The water-soluble receptor of this permease, HisJ, binds L-histidine and L-arginine (tightly) and L-lysine and L-ornithine (less tightly) in the periplasm, interacts with the membrane-bound complex (HisQMP2) and induces its ATPase activity, which results in ligand translocation. HisJ is a two-domain protein; in the absence of ligand, the cleft between two domains is open and binding of substrate stabilizes the closed conformation. Surprisingly, various liganded HisJ forms display substantial differences in their physicochemical characteristics and capacity to induce the ATPase. This is due to either different effects of the individual ligands on the respective closed structures, or to different equilibria being reached for each ligand between the open liganded form and the closed liganded form [Wolf, A. , Lee, K.C., Kirsch, J.F. & Ames, G.F.-L. (1996) J. Biol. Chem. 271, 21243-21250]. In this work, time-resolved measurements of the decay of intrinsic HisJ fluorescence and of the decay of the anisotropy of the fluorescence, as well as the analysis of the steady-state near UV CD and fluorescence spectra, rule out the model in which the differences between liganded complexes reflect different equilibria. The decay of the anisotropy of the fluorescence shows that liganded complexes differ dramatically in their large-scale conformational dynamics. Differential scanning calorimetry (DSC) curves for the HisJ thermal unfolding are well described by a scheme of equilibrium two-state unfolding of two independent domains, which can be ascribed to the two-domain structure of HisJ. This is true both for apo-HisJ at various pH values, and for HisJ in the presence of its ligands at varying concentrations, at pH 8.3. The DSC and structural data suggest that all ligands interact more extensively with the larger domain. A qualitative model for the HisJ conformational dynamics employing the idea of a twisting movement of the domains is proposed, which explains the difference in the efficacy of the ATPase induction by the various liganded HisJ forms.

Conformational flexibility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: implications for lipid binding.

An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.

Structure of a monoclonal 2E8 Fab antibody fragment specific for the low-density lipoprotein-receptor binding region of apolipoprotein E refined at 1.9 A.

The crystal structure of the Fab fragment of 2E8, the monoclonal IgG1,kappa antibody specific for the low-density lipoprotein (LDL) receptor-binding region of apolipoprotein E (apoE), has been solved by molecular replacement and refined at 1.9 A resolution (PDB entry 12E8). Two 2E8 Fab molecules in the asymmetric unit are related by noncrystallographic symmetry and are hydrogen bonded through a beta-sheet-like intermolecular contact between the heavy-chain complementarity-determining regions 3 (CDRH3) of each molecule. The structure has been refined to an R value of 0.22 (Rfree = 0.27). The initially ill-defined heavy-chain constant domain (CH1) of 2E8 has been retraced with the aid of automatic refinement, confirming the beta-sheet tracing independently of any starting models. As a resolution better than 2 A is not common for Fab fragments, this model represents a well defined Fab structure and should prove useful in MR solution of other Fab fragments. Furthermore, in the absence of an LDL-receptor structure, the homology of the 2E8 CDRH2 to the ligand-binding domain of the LDL receptor has been exploited to model the apoE-LDL-receptor interaction.

Cadmium-induced crystallization of proteins: II. Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine.

To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine-binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine-binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range. In each case, crystals grown in the presence of cadmium diffract to better than 2.0 angstroms resolution and belong to the orthorhombic space group P2(1)2(1)2(1). From our results and from the analysis of cadmium sites in well-refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.

Novel mechanism for defective receptor binding of apolipoprotein E2 in type III hyperlipoproteinemia.

The defective binding of apolipoprotein (apo) E2 to lipoprotein receptors, an underlying cause of type III hyperlipoproteinemia, results from replacement of Arg 158 with Cys, disrupting the naturally occurring salt bridge between Asp 154 and Arg 158. A new bond between Asp 154 and Arg 150 is formed, shifting Arg 150 out of the receptor binding region. Elimination of the 154-150 salt bridge by site-directed mutagenesis of Asp 154 to Ala restored the receptor binding activity to near normal levels. The X-ray crystal structure of apoE2 Ala 154 demonstrated that Arg 150 was relocated within the receptor binding region. Our results demonstrate that defective binding of apoE2 occurs by a novel mechanism of the replacement of one salt bridge with another.

2 A resolution structure of DppA, a periplasmic dipeptide transport/chemosensory receptor.

The family of about 50 periplasmic binding proteins, which exhibit diverse specificity (e.g., carbohydrates, amino acids, dipeptides, oligopeptides, oxyanions, metals, and vitamins) and range in size from 20 to 58 kDa, is a gold mine for an atomic-level investigation of structure and molecular recognition. These proteins serve as initial receptors for active transport systems or permeases. About six of these proteins, including the dipeptide-binding protein (DppA), are also primary receptors for chemotaxis. The structure of the unbound form of DppA (M(r) = 57,400) has been determined and refined to an R-factor of 0.169 to 2 A resolution. DppA consists of two distinct domains (I and II) connected by two "hinge" segments which form part of the base of the wide groove between the two domains. The relative orientation of the two domains gives the protein a pearlike shape, with domain I and domain II forming the larger and smaller apical ends, respectively. From the tip to the rounded bottom measures about 85 A, and the widest diameter is about 60 A. Domain I, which consists of two integrated subdomains, is folded from two separate polypeptide segments from the amino- and carboxyl-terminal ends. The more compact domain II is formed from the intervening segment. Comparison of the dipeptide-binding protein structure with that of the bound form of the similar oligopeptide-binding protein [Tame, J. R. H., Murshudov, G. N., Dodson, E. J., Neil, T. K., Dodson, G. G., Higgins, C. F., & Wilkinson, A. J. (1994) Science 264, 1578-1581] reveals the major features that differentiate the ligand specificity of the two proteins and describe the large hinge bending (about 55 degrees) between the two domains.

Influence of divalent cations in protein crystallization.

We have tested the effect of several cations in attempts to crystallize the ligand-bound forms of the leucine/isoleucine/valine-binding protein (LIVBP) (M(r) = 36,700) and leucine-specific binding protein (LBP) (M(r) = 37,000), which act as initial periplasmic receptors for the high-affinity osmotic-shock-sensitive active transport system in bacterial cells. Success was achieved with Cd2+ promoting the most dramatic improvement in crystal size, morphology, and diffraction quality. This comes about 15 years after the ligand-free proteins were crystallized. Nine other different divalent cations were tried as additives in the crystallization of LIVBP with polyethylene glycol 8000 as precipitant, and each showed different effects on the crystal quality and morphology. Cd2+ produced large hexagonal prism crystals of LIVBP, whereas a majority of the cations resulted in less desirable needle-shaped crystals. Zn2+ gave crystals that are long rods with hexagonal cross sections, a shape intermediate between the hexagonal prism and needle forms. The concentration of Cd2+ is critical. The best crystals of the LIVBP were obtained in the presence of 1 mM CdCl2, whereas those of LBP, with trigonal prism morphology, were obtained at a much higher concentration of 100 mM. Both crystals diffract to at least 1.7 A resolution using a conventional X-ray source.

Refined 1.89-A structure of the histidine-binding protein complexed with histidine and its relationship with many other active transport/chemosensory proteins.

The structure of the histidine-binding protein (HBP, M(r) = 26,100), involved solely in active transport, has been determined by the molecular replacement technique and refined to 1.89-A resolution and to an R-factor of 0.199. The structure is that of two protein molecules, each with a bound L-histidine, in the asymmetric unit. Replacement solution was achieved by using a model of the crystal structure of the ligand-free, open-cleft form of the lysine/arginine/ornithine-binding protein which was modified so that the two domains are close to each other by bending the hinge connecting the two domains. The bound histidine is held in place by 10 hydrogen bonds, 2 salt links, and about 60 van der Waals contacts. Elucidation of the HBP structure brings a total of eight different binding proteins structures determined in our laboratory, including those with specificities for monosaccharides, maltodextrins (linear and cyclic), aliphatic amino acids, and inorganic oxyanions. These structures comprise about a third of the entire family of periplasmic binding proteins which act as initial primary high-affinity receptors of active transport in Gram-negative bacteria. Two of the binding proteins with specificities for glucose/galactose and maltodextrins also serve in a similar capacity in chemotaxis. Though these proteins have different molecular weights (ranging from 26,000 to 40,000), amino acid sequences, and ligand specificities, their three-dimensional structures are similar overall. They are elongated (axial ratios of 2:1) and composed of two similar globular domains separated by a deep cleft wherein the ligand-binding site is located. These structures provide understanding of molecular recognition of a variety of ligands at the atomic level and functional roles of the binding proteins.

Leucine/isoleucine/valine-binding protein contracts upon binding of ligand.

Small-angle x-ray scattering and computer modeling have been used to study the effects of ligand binding to the leucine/isoleucine/valine-binding protein, an initial component of the high-affinity active transport system for branched-chain aliphatic amino acids in Escherichia coli. Measurements were made with no ligand present and with either L-leucine or L-valine present. Upon binding of either leucine or valine, there is a decrease in the radius of gyration, from 23.2 +/- 0.2 to 22.2 +/- 0.2 A, and in the maximum particle dimension, from 82 +/- 3 to 73 +/- 3 A. The x-ray structure of the unbound form has been determined and gives a radius of gyration and a maximum dimension consistent with the values found for the solution structure in this study (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). The reduction in the radius of gyration and maximum dimension upon ligand binding can be accounted for by a substrate-induced cleft closure in a combined "hinge-twist" motion. Modeling of the substrate-bound state was done by comparison of this protein with another periplasmic binding protein (L-arabinose-binding protein), which possesses a similar two-lobe structure and for which the x-ray structure is known in its ligand-bound form.

[Spatial structure of the basic chain of the neurotoxin I molecule from the venom of cobra Naja naja oxiana and its crystal packing]. / Prostranstvennoe stroenie osnovnoi tsepi molekuly neirotoksina I iz iada kobry Naja naja oxiana i ee ukladka v kristallicheskoi iacheike.

The structure of the alpha-carbon chain was solved by molecular replacement method at 2.7 A resolution. Neurotoxin I (NTX-I) is one of the main protein components purified from the venom of the central asian cobra Naja naja oxiana. NTX-I is known to bind specifically to acetylcholine receptors thus preventing the transmission of the neuroconductivity signal from synapses to muscles. NTX-I crystals were grown either by vapour diffusion or dialysis methods using specially prepared microdialysis cell. The intensities of reflections from native NTX-I crystals were measured in the range of 38.0-2.1 A-1 by omega-scan method with a Syntex P21 diffractometer operated in automatic regime. To determine the position and mode of packing of NTX-I molecule in unit cell program packages MERLOT and BLANC were applied running on a NORD-500 computer.

Crystallization and preliminary x-ray diffraction study of neurotoxin-I from Naja naja oxiana venom.

Crystals of the neurotoxin-I (NTX-I) from the venom of the middle Asian cobra Naja naja oxiana have been grown by vapour diffusion and dialysis methods. The crystals belong to space group P2(1)2(1)2 with dimension of a = 25.19 A, b = 75.59 A, c = 36.09 A and diffract to 1.9 A resolution. The asymmetric unit contains one molecule (Vm = 2.2 A/Da). Using the molecule of alpha-cobratoxin (CTX) as a starting model for NTX-I structure determination coordinates of C alpha atoms of the NTX-I molecule were obtained and the position of NTX-I in the unit cell was derived.

Preliminary X-ray investigation of 70 S ribosome crystals from Thermus thermophilus.

Large three-dimensional crystals of 70 S from Thermus thermophilus have been grown from solutions of 2-methyl-2,4-pentanediol at 4 degrees C and examined in an X-ray synchrotron beam. The space group is P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = 510 A and c = 378 A. The diffraction patterns extend to better than 20 A.

[Modification of a microdialysis method for crystallization of proteins]. / Modifikatsii mikrodializnogo metoda kristallizatsii belkov.

Several variants of microdialysis cells have been developed for growing protein crystals suitable for X-ray analysis. The cells have simple construction and are made of easily available materials. Using these cells, over ten proteins have been crystallized, six of them in the form suitable for 3D-structure determination.

Crystallization and preliminary X-ray crystallographic data of a histidine-binding protein from Escherichia coli.

Histidine-binding protein, purified from periplasmic space of Escherichia coli K12, has been crystallized in a form suitable for X-ray analysis. Crystals of average size 0.3 mm x 0.15 mm x 0.15 mm have been grown by the hanging-drop method, with ammonium sulfate as precipitant. The space group if I4(1)22, with the unit cell dimensions a = b = 119.1 A; c = 151.8 A; Vm = 2.7 A3/dalton. There appear to be two protein subunits of molecular weight 25,000 each in the asymmetric unit.

Structure of the L-leucine-binding protein refined at 2.4 A resolution and comparison with the Leu/Ile/Val-binding protein structure.

The three-dimensional X-ray structure of the leucine-binding protein (36,900 Mr and 346 residues), an active transport component of Escherichia coli, has been determined by the method of molecular replacement, using the refined structure of the Leu/Ile/Val-binding protein (344 residues) as the model structure. The two amino acid-binding proteins have 80% sequence identity and, although both crystallize in the same space group, they have very different unit cell dimensions. The rotation function yielded one significant peak, which subsequently led to a single self-consistent translation function solution. The model was first refined by the constrained least-squares method, with each of the two domains of the molecule treated separately to allow for any small change in the relative orientation of the two domains. The model was then modified in order to reflect the 72 changes in amino acid side-chains and two insertions in going from the Leu/Ile/Val-binding protein sequence to that of the L-leucine-binding protein. Final structure refinement, using the restrained least-squares technique, resulted in an R-factor of 0.20 for 13,797 reflections to a resolution of 2.4 A. The model is comprised of 2600 protein atoms and 91 solvent molecules. The L-leucine-binding protein structure is, as expected, very similar to the Leu/Ile/Val-binding protein structure; both are in the unliganded conformation with the cleft between the two domains wide open and easily accessible. The superimposing of the structures yields a root-mean-square difference of 0.68 A in the alpha-carbon atoms of the 317 equivalent residues. The five regions of the leucine-binding protein structure that differ by more than 1.6 A from the Leu/Ile/Val-binding protein structure are far from the major portion of the ligand-binding site, which is located in one domain of the bilobate protein. Between the structures, there are three differences in the amino acid side-chains that form the major portion of the substrate-binding sites. These substitutions, by themselves, fail to clearly explain the differences in the specificities for branched aliphatic amino acids.