The interdependence of protein surface topography and bound water molecules revealed by surface accessibility and fractal density measures.

To characterize water binding to proteins, which is fundamental to protein folding, stability and activity, the relationships of 10,837 bound water positions to protein surface shape and residue type were analyzed in 56 high-resolution crystallographic structures. Fractal atomic density and accessibility algorithms provided an objective characterization of deep grooves in solvent-accessible protein surfaces. These deep grooves consistently had approximately the diameter of one water molecule, suggesting that deep grooves are formed by the interactions between protein atoms and bound water molecules. Protein surface topography dominates the chemistry and extent of water binding. Protein surface area within grooves bound three times as many water molecules as non-groove surface; grooves accounted for one-quarter of the total surface area yet bound half the water molecules. Moreover, only within grooves did bound water molecules discriminate between different side-chains. In grooves, main-chain surface was as hydrated as that of the most hydrophilic side-chains, Asp and Glu, whereas outside grooves all main and side-chains bound water to a similar, and much decreased, extent. This identification of the interdependence of protein surface shape and hydration has general implications for modelling and prediction of protein surface shape, recognition, local folding and solvent binding.     

Reassessing buried surface areas in protein–protein complexes

The buried surface area (BSA), which measures the size of the interface in a protein–protein complex may differ from the accessible surface area (ASA) lost upon association (which we call DSA), if conformation changes take place. To evaluate the DSA, we measure the ASA of the interface atoms in the bound and unbound states of the components of 144 protein–protein complexes taken from the Protein–Protein Interaction Affinity Database of Kastritis et al. (2011). We observe differences exceeding 20%, and a systematic bias in the distribution. On average, the ASA calculated in the bound state of the components is 3.3% greater than in their unbound state, and the BSA, 7% greater than the DSA. The bias is observed even in complexes where the conformation changes are small. An examination of the bound and unbound structures points to a possible origin: local movements optimize contacts with the other component at the cost of internal contacts, and presumably also the binding free energy.     

X-ray structure refinement and comparison of three forms of mitochondrial aspartate aminotransferase.

The X-ray crystal structures of three forms of the enzyme aspartate aminotransferase (EC 2.6.1.1) from chicken heart mitochondria have been refined by least-squares methods: holoenzyme with the co-factor pyridoxal-5'-phosphate bound at pH 7.5 (1.9 A resolution), holoenzyme with pyridoxal-5'-phosphate bound at pH 5.1 (2.3 A resolution) and holoenzyme with the co-factor pyridoxamine-5'-phosphate bound at pH 7.5 (2.2 A resolution). The crystallographic agreement factors [formula: see text] for the structures are 0.166, 0.130 and 0.131, respectively, for all data in the resolution range from 10.0 A to the limit of diffraction for each structure. The secondary, super-secondary and domain structures of the pyridoxal-phosphate holoenzyme at pH 7.5 are described in detail. The surface area of the interface between the monomer subunits of this dimeric alpha 2 protein is unusually large, indicating a very stable dimer. This is consistent with biochemical data. Both subunit and domain interfaces are relatively smooth compared with other proteins. The interactions of the protein with its co-factor are described and compared among the three structures. Observed changes in co-factor conformation may be related to spectral changes and the energetics of the catalytic reaction. Small but significant adjustments of the protein to changes in co-factor conformation are seen. These adjustments may be accommodated by small rigid-body shifts of secondary structural elements, and by packing defects in the protein core.     

Mammalian sperm-egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion.

Rat epididymal protein DE is localized on the fusogenic region of the acrosome-reacted spermatozoa and has a potential role in sperm-egg fusion. We investigated the presence of DE binding sites on the egg surface by co-incubating zona-free eggs and capacitated sperm in different concentrations of pure DE. Results indicate that DE produced a concentration-dependent decrease in egg penetration by sperm (fusion), with almost complete inhibition at 200 micrograms/ml. This inhibition was not due to an effect of DE on initial sperm binding to the egg membrane, since the presence of this protein did not affect the percentage of oocytes with bound sperm nor the number of bound sperm per egg. Those sperm that failed to penetrate the egg in the presence of DE became able to do so after transfer of the eggs to protein- and sperm-free medium, indicating a role for DE in an event subsequent to binding and leading to fusion. Indirect immunofluorescence using a polyclonal antibody against DE revealed a patchy labeling over the entire egg surface, with the exception of the area overlying the second metaphase spindle. This conclusion was supported by the disappearance of the DE-negative area on the fertilized egg. Zona-free eggs, incubated with DE at 4 degrees C or fixed before exposure to DE, displayed a uniform staining, suggesting that the patchy labeling resulted from aggregation of DE binding sites by the purified protein. The aggregation of these egg components may represent a necessary step of the fusion process. To our knowledge, this is the first study reporting the existence and localization of complementary sites to a specific sperm protein on the plasma membrane of the mammalian egg.     

Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure.

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.     

Tyrosine phosphorylation and its possible role in superoxide production by human neutrophils stimulated with FMLP and IgG.

Superoxide production by human neutrophils stimulated with FMLP and soluble aggregated human IgG were inhibited in a dose dependent manner by two kinds of tyrosine kinase inhibitors, erbstatin and genistein. Superoxide production stimulated with surface bound IgG, however, was scarcely inhibited by either inhibitor. Protein tyrosine phosphorylation studies with immunoblotting revealed specific tyrosine phosphorylation of a 40 Kd protein by soluble aggregated and surface bound IgG, and that of a 39 Kd protein, as well as the 40 Kd protein, by FMLP. These were all inhibited by the tyrosine kinase inhibitors. These data suggest that superoxide production induced by FMLP and soluble aggregated IgG are, at least in part, tyrosine kinase dependent, but the tyrosine kinases and/or substrates of tyrosine kinases involved may be different. In addition, tyrosine kinase independent pathways are also suggested to be involved in superoxide production by stimulation with surface bound IgG.     

Reversible self-association of ovalbumin at air-water interfaces and the consequences for the exerted surface pressure

In this study the relation between the ability of protein self-association and the surface properties at air-water interfaces is investigated using a combination of spectroscopic techniques. Three forms of chicken egg ovalbumin were obtained with different self-associating behavior: native ovalbumin, heat-treated ov-albumin-being a cluster of 12-16 predominantly noncovalently bound proteins, and succinylated ovalbumin, as a form with diminished aggregation properties due to increased electrostatic repulsion. While the bulk diffusion of aggregated protein is clearly slower compared to monomeric protein, the efficiency of transport to the interface is increased, just like the efficiency of sticking to rather than bouncing from the interface. On a timescale of hours, the aggregated protein dissociates and adopts a conformation comparable to that of native protein adsorbed to the interface. The exerted surface pressure is higher for aggregated material, most probably because the deformability of the particle is smaller. Aggregated protein has a lower ability to desorb from the interface upon compression of the surface layer, resulting in a steadily increasing surface pressure upon reducing the available area for the surface layer. This observation is opposite to what is observed for succinylated protein that may desorb more easily and thereby suppresses the buildup of a surface pressure. Generally, this work demonstrates that modulating the ability of proteins to self-associate offers a tool to control the rheological properties of interfaces.     

How a G protein binds a membrane

Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated Ggamma C terminus and Galpha N terminus, and another near the Galpha C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions.     

Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy.

The interaction of a nonspecific wheat lipid transfer protein (LTP) with phospholipids has been studied using the monolayer technique as a simplified model of biological membranes. The molecular organization of the LTP-phospholipid monolayer has been determined by using polarized attenuated total internal reflectance infrared spectroscopy, and detailed information on the microstructure of the mixed films has been investigated by using epifluorescence microscopy. The results show that the incorporation of wheat LTP within the lipid monolayers is surface-pressure dependent. When LTP is injected into the subphase under a dipalmytoylphosphatidylglycerol monolayer at low surface pressure (< 20 mN/m), insertion of the protein within the lipid monolayer leads to an expansion of dipalmytoylphosphatidylglycerol surface area. This incorporation leads to a decrease in the conformational order of the lipid acyl chains and results in an increase in the size of the solid lipid domains, suggesting that LTP penetrates both expanded and solid domains. By contrast, when the protein is injected under the lipid at high surface pressure (> or = 20 mN/m) the presence of LTP leads neither to an increase of molecular area nor to a change of the lipid order, even though some protein molecules are bound to the surface of the monolayer, which leads to an increase of the exposure of the lipid ester groups to the aqueous environment. On the other hand, the conformation of LTP, as well as the orientation of alpha-helices, is surface-pressure dependent. At low surface pressure, the alpha-helices inserted into the monolayers are rather parallel to the monolayer plane. In contrast, at high surface pressure, the alpha-helices bound to the surface of the monolayers are neither parallel nor perpendicular to the interface but in an oblique orientation.     

The physiological structure of human C-reactive protein and its complex with phosphocholine.

BACKGROUND: Human C-reactive protein (CRP) is the classical acute phase reactant, the circulating concentration of which rises rapidly and extensively in a cytokine-mediated response to tissue injury, infection and inflammation. Serum CRP values are routinely measured, empirically, to detect and monitor many human diseases. However, CRP is likely to have important host defence, scavenging and metabolic functions through its capacity for calcium-dependent binding to exogenous and autologous molecules containing phosphocholine (PC) and then activating the classical complement pathway. CRP may also have pathogenic effects and the recent discovery of a prognostic association between increased CRP production and coronary atherothrombotic events is of particular interest. RESUTLS: The X-ray structures of fully calcified C-reactive protein, in the presence and absence of bound PC, reveal that although the subunit beta-sheet jellyroll fold is very similar to that of the homologous pentameric protein serum amyloid P component, each subunit is tipped towards the fivefold axis. PC is bound in a shallow surface pocket on each subunit, interacting with the two protein-bound calcium ions via the phosphate group and with Glu81 via the choline moiety. There is also an unexpected hydrophobic pocket adjacent to the ligand. CONCLUSIONS: The structure shows how large ligands containing PC may be bound by CRP via a phosphate oxygen that projects away from the surface of the protein. Multipoint attachment of one planar face of the CRP molecule to a PC-bearing surface would leave available, on the opposite exposed face, the recognition sites for C1q, which have been identified by mutagenesis. This would enable CRP to target physiologically and/or pathologically significant complement activation. The hydrophobic pocket adjacent to bound PC invites the design of inhibitors of CRP binding that may have therapeutic relevance to the possible role of CRP in atherothrombotic events.     

WaterScore: a novel method for distinguishing between bound and displaceable water molecules in the crystal structure of the binding site of protein-ligand complexes

We have performed a multivariate logistic regression analysis to establish a statistical correlation between the structural properties of water molecules in the binding site of a free protein crystal structure, with the probability of observing the water molecules in the same location in the crystal structure of the ligand-complexed form. The temperature B-factor, the solvent-contact surface area, the total hydrogen bond energy and the number of protein-water contacts were found to discriminate between bound and displaceable water molecules in the best regression functions obtained. These functions may be used to identify those bound water molecules that should be included in structure-based drug design and ligand docking algorithms.FIGURE The binding site ( thin sticks) of penicillopepsin (3app) with its crystallographically determined water molecules ( spheres) and superimposed ligand (in thick sticks, from complexed structure 1ppk). Water molecules sterically displaced by the ligand upon complexation are shown in cyan. Bound water molecules are shown in blue. Displaced water molecules are shown in yellow. Water molecules removed from the analysis due to a lack of hydrogen bonds to the protein are shown in white. WaterScore correctly predicted waters in blue as Probability=1 to remain bound and waters in yellow as Probability<1x10(-20) to remain bound.     

Thermodynamic analysis of the effect of concentrated salts on protein interaction with hydrophobic and polysaccharide columns

An attempt was made to explain the effect of concentrated salts on protein interaction with hydrophobic columns. From the previously observed results of preferential interactions for salting-out salts with proteins, it was shown that the free energy of the protein is increased by addition of the salts and this unfavorable free energy is smaller for the proteins bound to the columns because of their smaller surface area exposed to solvent; i.e., the bound form of the proteins is thermodynamically more stable. This explains the protein binding to the hydrophobic columns at high salt concentrations and the elution by decreasing the salt concentration. The unfavorable interaction free energy was greater for Na2SO4 or (NH4)2SO4 than for NaCl, which explains the stronger effect of the former salts on the protein binding to the columns. The observed favorable interaction between KSCN or guanidine hydrochloride and the proteins explains the decreasing effect of these salts on the protein binding to the hydrophobic columns.     

A 60-kD protein mediates the binding of transforming growth factor-beta to cell surface and extracellular matrix proteoglycans

The biological activity of many cytokines is regulated by binding proteins present at the cell surface, in extracellular matrices or in soluble phase. We describe here a TGF-beta binding protein that is both an extracellular matrix and a cell surface protein. When intact extracellular matrices of HEP-G2 cells were affinity cross-linked with 125I-TGF-beta 1, two major binding components were seen: a 250-kD, proteoglycan-like molecule, presumed to be betaglycan, and a 60-kD protein. The 60-kD TGF-beta-binding protein was also present at the cell surface. It could be released from the cell surface by treating cells with high salt, heparin, chondroitin sulfate, heparitinase, or chondroitinase, indicating that it is bound to heparan sulfate and chondroitin sulfate proteoglycans. The 60-kD protein bound TGF-beta 1 with an apparent dissociation constant of 1.6 nM, and there were 30,000 binding sites per cell at the cell surface. In addition to the HEP-G2 cells and another hepatoma cell line, the 60-kD protein was also found in a human colon carcinoma (HT-29) cell line but not in rat kidney (NRK- 49F) or human fibroblast (HUT-12) cell lines. The 60-kD protein could be extracted from cells containing it and transferred to the surface of previously negative cells. The 60-kD protein may serve to regulate the binding of TGF-beta to its signal transducing receptors by targeting TGF-beta to appropriate locations in the microenvironment of cells.     

Neutrophil activation by surface bound IgG is via a pertussis toxin insensitive G protein

Pre-treatment of neutrophils with either pertussis or cholera toxins does not inhibit neutrophil activation by surface bound IgG. In contrast, pretreatment with the phorbol ester, phorbol myristate acetate, results in a dose dependent inhibition of degranulation by surface bound IgG. This inhibition is similar to that seen with soluble ligands where it is thought to be due to interference with the interaction of an activated guanine nucleotide binding protein with phospholipase C (J. Biol. Chem.,262,6121,1987). More directly, GTP binding and GTPase activity are enhanced when human neutrophil membranes are incubated in wells containing surface bound IgG. Neither of these G protein functions were inhibited when membranes were prepared in the presence of pertussis toxin, suggesting that neutrophil activation by surface bound IgG proceeds by a mechanism that involves a pertussis toxin insensitive G protein.     

Structural evidence for a dominant role of nonpolar interactions in the binding of a transport/chemosensory receptor to its highly polar ligands.

The receptor, a maltose/maltooligosaccharide-binding protein, has been found to be an excellent system for the study of molecular recognition because its polar and nonpolar binding functions are segregated into two globular domains. The X-ray structures of the "closed" and "open" forms of the protein complexed with maltose and maltotetraitol have been determined. These sugars have approximately 3 times more accessible polar surface (from OH groups) than nonpolar surface (from small clusters of sugar ring CH bonds). In the closed structures, the oligosaccharides are buried in the groove between the two domains of the protein and bound by extensive hydrogen bonding interactions of the OH groups with the polar residues confined mostly in one domain and by nonpolar interactions of the CH clusters with four aromatic residues lodged in the other domain. Substantial contacts between the sugar hydroxyls and aromatic residues are also formed. In the open structures, the oligosaccharides are bound almost exclusively in the domain rich in aromatic residues. This finding, along with the analysis of buried surface area due to complex formations in the open and closed structures, supports a major role for nonpolar interactions in initial ligand binding even when the ligands have significantly greater potential for highly specific polar interactions.     

Relative solvent accessible surface area predicts protein conformational changes upon binding

Protein interactions are often accompanied by significant changes in conformation. We have analyzed the relationships between protein structures and the conformational changes they undergo upon binding. Based upon this, we introduce a simple measure, the relative solvent accessible surface area, which can be used to predict the magnitude of binding-induced conformational changes from the structures of either monomeric proteins or bound subunits. Applying this to a large set of protein complexes suggests that large conformational changes upon binding are common. In addition, we observe considerable enrichment of intrinsically disordered sequences in proteins predicted to undergo large conformational changes. Finally, we demonstrate that the relative solvent accessible surface area of monomeric proteins can be used as a simple proxy for protein flexibility. This reveals a powerful connection between the flexibility of unbound proteins and their binding-induced conformational changes, consistent with the conformational selection model of molecular recognition.     

Linkage of EcoRI dissociation from its specific DNA recognition site to water activity, salt concentration, and pH: separating their roles in specific and non-specific binding.

We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.     

2-Step purification of the Ku DNA repair protein expressed in Escherichia coli

The Ku protein is involved in DNA double-strand break repair by non-homologous end-joining (NHEJ), which is crucial to the maintenance of genomic integrity in mammals. To study the role of Ku in NHEJ we developed a bicistronic Escherichia coli expression system for the Ku70 and Ku80 subunits. Association of the Ku70 and Ku80 subunits buries a substantial amount of surface area (approximately 9000 A2 [J.R. Walker, R.A. Corpina, J. Goldberg, Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair, Nature 412 (2001) 607-614]), which suggests that herterodimerization may be important for protein stability. N-terminally His6-tagged Ku80 was soluble in the presence, but not in the absence, of bicistronically expressed untagged Ku70. In a 2-step purification, metal chelating affinity chromatography was followed by step-gradient elution from heparin-agarose. Co-purification of equimolar amounts of His6-tagged Ku80 and untagged Ku70 was observed, which indicated heterodimerization. Recombinant Ku bound dsDNA, activated the catalytic subunit of the DNA-dependent kinase (DNA-PKcs) and functioned in NHEJ reactions in vitro. Our results demonstrate that while the heterodimeric interface of Ku is extensive it is nonetheless possible to produce biologically active Ku protein in E. coli.     

Protein adsorption on core-shell resins for flow-through purifications: Effect of protein molecular size,shape, and salt concentration

This work addresses the functional properties of the core-shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose-based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.