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.     

Computer programs to identify and classify amphipathic alpha helical domains.

The amphipathic alpha helix is an often-encountered secondary structural motif in biologically active peptides and proteins. An amphipathic helix is defined as an alpha helix with opposing polar and nonpolar faces oriented along the long axis of the helix. In a recent review article we grouped amphipathic helixes into seven distinct classes (A, H, L, G, K, C, and M) based upon a detailed analysis of their physical-chemical and structural properties (Segrest, J. P., et al. Amphipathic helix motif: classes and properties. Proteins. 1990. 8: 103-117). We have developed five computer programs that automate analysis and classification of potential amphipathic helical domains from primary amino acid sequence data. Here we describe these five programs and illustrate their usefulness by comparing two data sets of sequences representing different amphipathic alpha helical motifs from the exchangeable apolipoproteins. In a companion review article (Segrest, J. P., et al. The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function. J. Lipid Res. 1992. 33: 000-000) these five programs are used to localize and characterize the putative amphipathic helixes in the exchangeable apolipoproteins.     

A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein

The E1 glycoprotein from an avian coronavirus is a model protein for studying retention in the Golgi complex. In animal cells expressing the protein from cDNA, the E1 protein is targeted to cis Golgi cisternae (Machamer, C. E., S. A. Mentone, J. K. Rose, and M. G. Farquhar. 1990. Proc. Natl. Acad. Sci. USA. 87:6944-6948). We show that the first of the three membrane-spanning domains of the E1 protein can retain two different plasma membrane proteins in the Golgi region of transfected cells. Both the vesicular stomatitis virus G protein and the alpha-subunit of human chorionic gonadotropin (anchored to the membrane by fusion with the G protein membrane-spanning domain and cytoplasmic tail) were retained in the Golgi region of transfected cells when their single membrane-spanning domains were replaced with the first membrane-spanning domain from E1. Single amino acid substitutions in this sequence released retention of the chimeric G protein, as well as a mutant E1 protein which lacks the second and third membrane-spanning domains. The important feature of the retention sequence appears to be the uncharged polar residues which line one face of a predicted alpha helix. This is the first retention signal to be defined for a resident Golgi protein. The fact that it is present in a membrane-spanning domain suggests a novel mechanism of retention in which the membrane composition of the Golgi complex plays an instrumental role in retaining its resident proteins.     

Molecular dynamics study of time-correlated protein domain motions and molecular flexibility: cytochrome P450BM-3.

Time-correlated atomic motions were used to characterize protein domain boundaries from atomic coordinates generated by molecular dynamics simulations. A novel application of the dynamical cross-correlation matrix (DCCM) analysis tool was used to help identify putative protein domains. In implementing this new approach, several DCCM maps were calculated, each using a different coordinate reference frame from which protein domain boundaries and protein domain residue constituents could be identified. Cytochrome P450BM-3, from Bacillus megaterium, was used as the model protein in this study. The analyses indicated that the simulated protein comprises three distinct domain regions; in contrast, only two protein domains were identified in the original crystal structure report. Specifically, the DCCM analyses showed that the F-G helix region was a separate domain entity and not a part of the alpha domain, as previously designated. The simulations demonstrated that the domain motions of the F-G helix region effected both the size and shape of the enzyme active site, and that the dynamics of the F-G helix domain could possibly control access of substrate to the binding pocket.     

S111N mutation in the helical domain of human Gs(alpha) reduces its GDP/GTP exchange rate

G-protein alpha subunits consist of two domains: a Ras-like domain also called GTPase domain (GTPaseD), structurally homologous to monomeric G-proteins, and a more divergent domain, unique to heterotrimeric G-proteins, called helical domain (HD). G-protein activation, requires the exchange of bound GDP for GTP, and since the guanine nucleotide is buried in a deep cleft between both domains, it has been postulated that activation may involve a conformational change that will allow the opening of this cleft. Therefore, it has been proposed, that interdomain interactions are playing an important role in regulating the nucleotide exchange rate of the alpha subunit. While constructing different Gs(alpha) quimeras, we identified a Gs(alpha) random mutant, which was very inefficient in stimulating adenylyl cyclase activity. The introduced mutation corresponded to the substitution of Ser(111) for Asn (S111N), located in the carboxi terminal end of helix A of the HD, a region neither involved in AC interaction nor in the interdomain interface. In order to characterize this mutant, we expressed it in bacteria, purified it by niquel-agarose chromatography, and studied its nucleotide exchange properties. We demonstrated that the recombinant S111N Gs(alpha) was functional since it was able to undergo the characteristic conformational change upon GTP binding, detected by the acquisition of a trypsin-resistant conformation. When the biochemical properties were determined, the mutant protein exhibited a reduced GDP dissociation kinetics and as a consequence a slower GTPgammaS binding rate that was responsible for a diminished adenylyl cyclase activation when GTPgammaS was used as activator. These data provide new evidence that involves the HD as a regulator of Gs(alpha) function, in this case the alphaA helix, which is not directly involved with the nucleotide binding site nor the interdomain interface.     

Stabilizing nonpolar/polar side-chain interactions in the alpha-helix.

A simplistic, yet often used, view of protein stability is that amino acids attract other amino acids with similar polarity, whereas nonpolar and polar side chains repel. Here we show that nonpolar/polar interactions, namely Val or Ile bonding to Lys or Arg in alpha-helices, can in fact be stabilizing. Residues spaced i, i + 4 in alpha-helices are on the same face of the helix, with potential to favorably interact and stabilize the structure. We observe that the nonpolar/polar pairs Ile-Lys, Ile-Arg, and Val-Lys occur in protein helices more often than expected when spaced i, i + 4. Partially helical peptides containing pairs of nonpolar/polar residues were synthesized. Controls with i, i + 5 spacing have the residues on opposite faces of the helix and are less helical than the test peptides with the i, i + 4 interactions. Experimental circular dichroism results were analyzed with helix-coil theory to calculate the free energy for the interactions. All three stabilize the helix with DeltaG between -0.14 and -0.32 kcal x mol(-1). The interactions are hydrophobic with contacts between Val or Ile and the alkyl groups in Arg or Lys. Side chains such as Lys and Arg can thus interact favorably with both polar and nonpolar residues.     

The membrane association domain of RGS16 contains unique amphipathic features that are conserved in RGS4 and RGS5.

Regulators of G protein signaling (RGS proteins) modulate G protein-mediated signaling pathways by acting as GTPase-activating proteins for Gi, Gq, and G12 alpha-subunits of heterotrimeric G proteins. Although it is known that membrane association is critical for the biological activities of many RGS proteins, the mechanism underlying this requirement remains unclear. We reported recently that the NH2 terminus of RGS16 is required for its function in vivo. In this study, we show that RGS16 lacking the NH2 terminus is no longer localized to the plasma membrane as is the wild type protein, suggesting that membrane association is important for biological function. The region of amino acids 7-32 is sufficient to confer the membrane-targeting activity, of which amino acids 12-30 are predicted to adopt an amphipathic alpha-helix. Site-directed mutagenesis experiments showed that the hydrophobic residues of the nonpolar face of the helix and the strips of positively charged side chains positioned along the polar/nonpolar interface of the helix are crucial for membrane association. Subcellular fractionation by differential centrifugation followed by conditions that distinguish peripheral membrane proteins from integral ones indicate that RGS16 is a peripheral membrane protein. We show further that RGS16 membrane association does not require palmitoylation. Our results, together with other recent findings, have defined a unique membrane association domain with amphipathic features. We believe that these structural features and the mechanism of membrane association of RGS16 are likely to apply to the homologous domains in RGS4 and RGS5.     

The composition rather than position of polar residues (QxxS) drives aspartate receptor transmembrane domain dimerization in vivo

Transmembrane (TM) helix association is an important process affecting the function of many integral membrane proteins. Consequently, aberrations in this process are associated with diseases. Unfortunately, our knowledge of the factors that control this oligomerization process in the membrane milieu is limited at best. Previous studies have shown a role for polar residues in the assembly of synthetic peptides in vitro and the association of de novo-designed TM helices in vivo. Here we examined, for the first time, the involvement of polar residues in the dimerization of a biological TM domain in its natural environment. We analyzed both the involvement of polar residues in the dimerization process and whether their influence is position-dependent. For this purpose, we used the TM domain of the Escherichia coli aspartate receptor (Tar) and 10 single and double mutants. Polar to nonpolar mutations in the sequence demonstrated the role of the QxxS motif in the dimerization of the Tar TM domain. Moreover, creating a GxxxG motif, instead of the polar motif, almost completely abolished dimerization. Swapping positions between two wild-type polar residues did not affect dimerization, implying a similar contribution from both positions. Interestingly, mutants that contain two identical strong polar residues, EE and QQ, demonstrated a substantially higher level of dimerization than a QE mutant, although all three TM domains contain two strong polar residues. This result suggests that, in addition to the polarity of the residues, the formation of symmetric bonds also plays a role in dimer stability. The results of this study may facilitate a rational modulation of membrane protein function for therapeutic purposes.     

Interaction of beta2-glycoprotein 1 with phosphatidylserine-containing membranes: ligand-dependent conformational alterations initiate bivalent binding

Beta2-glycoprotein 1 (beta2GP1), a 50 kDa serum glycoprotein that binds anionic phospholipid-containing membranes, plays a regulatory role in physiology and pathology. The protein is a member of the short consensus repeat (SCR) superfamily containing four typical repeating domains and an aberrant fifth domain constructed into an SCR-like core at the C-terminus. To investigate the contribution of the individual domains to the binding of beta2GP1, a series of sequential domain-deleted recombinant protein fragments were generated and assessed for their interaction with PS-containing vesicles. Spectral analyses of lipid binding-dependent alterations in tryptophan emission spectra revealed that the (single) tryptophan residues of the individual domains underwent binding-dependent conformational alterations. Depending on the ionic strength, some domains moved from polar to nonpolar environments, while others moved from less polar to more polar environments. Analysis of a series of acrylamide quenching and resonance energy transfer experiments indicated that the binding of N-terminal domain 1 to PS membranes exists in two, ionic strength-dependent, conformations. At low ionic strengths, domain 1 bound to the vesicles and induced their precipitation and/or aggregation. At physiologic ionic strengths, domain 1 detached from the membrane surface while the remaining domains maintained their association with the membrane. Under these conditions, membrane-bound conformationally altered domain 1 projects away from the membrane surface, enabling it to interact with other proteins and/or cell surface ligands or receptors.     

Crosslinking between alpha and beta subunits defines the orientation and spatial relationship of some of the transmembrane helices of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli

The proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta, organized as an alpha(2)beta(2) tetramer. The protein contains three recognizable domains, of which domain II is the transmembrane region of the molecule containing the pathway for proton translocation. Domain II is composed of four transmembrane helices at the carboxyl-terminus of the alpha subunit and either eight or nine transmembrane helices at the amino-terminal region of the beta subunit. We have introduced pairs of cysteine residues into a cysteine-free transhydrogenase by site-directed mutagenesis. Disulfide bond formation between some of these cysteine residues occurred spontaneously or on treatment with cupric 1, 10-phenanthrolinate. Analysis of crosslinked products confirmed that there are nine transmembrane helices in the domain II region of the beta subunit. The proximity to one another of several of the transmembrane helices was determined. Thus, helices 2 and 4 are close to helix 6 (nomenclature of Meuller and Rydstr?m, J. Biol. Chem. 274, 19072-19080, 1999), and helix 3 and the carboxyl-terminal eight residues of the alpha subunit are close to helix 7. In the alpha(2)beta(2) tetramer, helices 2 and 4 of one alpha subunit are close to the same pair of transmembrane helices of the other alpha subunit, and helix 6 of one beta subunit is close to helix 6 of the other beta subunit.     

Structure and dynamics of the human pleckstrin DEP domain: distinct molecular features of a novel DEP domain subfamily

Pleckstrin1 is a major substrate for protein kinase C in platelets and leukocytes, and comprises a central DEP (disheveled, Egl-10, pleckstrin) domain, which is flanked by two PH (pleckstrin homology) domains. DEP domains display a unique alpha/beta fold and have been implicated in membrane binding utilizing different mechanisms. Using multiple sequence alignments and phylogenetic tree reconstructions, we find that 6 subfamilies of the DEP domain exist, of which pleckstrin represents a novel and distinct subfamily. To clarify structural determinants of the DEP fold and to gain further insight into the role of the DEP domain, we determined the three-dimensional structure of the pleckstrin DEP domain using heteronuclear NMR spectroscopy. Pleckstrin DEP shares main structural features with the DEP domains of disheveled and Epac, which belong to different DEP subfamilies. However, the pleckstrin DEP fold is distinct from these structures and contains an additional, short helix alpha4 inserted in the beta4-beta5 loop that exhibits increased backbone mobility as judged by NMR relaxation measurements. Based on sequence conservation, the helix alpha4 may also be present in the DEP domains of regulator of G-protein signaling (RGS) proteins, which are members of the same DEP subfamily. In pleckstrin, the DEP domain is surrounded by two PH domains. Structural analysis and charge complementarity suggest that the DEP domain may interact with the N-terminal PH domain in pleckstrin. Phosphorylation of the PH-DEP linker, which is required for pleckstrin function, could regulate such an intramolecular interaction. This suggests a role of the pleckstrin DEP domain in intramolecular domain interactions, which is distinct from the functions of other DEP domain subfamilies found so far.     

Domain orientation in the inactive response regulator Mycobacterium tuberculosis MtrA provides a barrier to activation

The structure of MtrA, an essential gene product for the human pathogen Mycobacterium tuberculosis, has been solved to a resolution of 2.1 A. MtrA is a member of the OmpR/PhoB family of response regulators and represents the fourth family member for which a structure of the protein in its inactive state has been determined. As is true for all OmpR/PhoB family members, MtrA possesses an N-terminal regulatory domain and a C-terminal winged helix-turn-helix DNA-binding domain, with phosphorylation of the regulatory domain modulating the activity of the protein. In the inactive form of MtrA, these two domains form an extensive interface that is composed of the alpha4-beta5-alpha5 face of the regulatory domain and the C-terminal end of the positioning helix, the trans-activation loop, and the recognition helix of the DNA-binding domain. This domain orientation suggests a mechanism of mutual inhibition by the two domains. Activation of MtrA would require a disruption of this interface to allow the alpha4-beta5-alpha5 face of the regulatory domain to form the intermolecule interactions that are associated with the active state and to allow the recognition helix to interact with DNA. Furthermore, the interface appears to stabilize the inactive conformation of MtrA, potentially reducing the rate of phosphorylation of the N-terminal domain. This combination of effects may form a switch, regulating the activity of MtrA. The domain orientation exhibited by MtrA also provides a rationale for the variation in linker length that is observed within the OmpR/PhoB family of response regulators.     

Lipid binding by fragments of apolipoprotein C-III-1 obtained by thrombin cleavage.

We have used thrombin to cleave apolipoprotein C-III-1 into two fragments constituting residues 1-40 (apoLP-C-III-A) and 41-79 (apoLP-C-III-B). The lipid binding properties of these fragments with dimyristoyl- and 1-palmitoyl-2-oleoylphosphatidylcholines have been determined using circular dichroic and intrinsic tryptophan fluorescence spectroscopy. The peptide-phospholipid mixtures were fractionated by density gradients of cesium chloride. ApoLP-C-III-A showed disordered structure in the absence and presence of DMPC and no significant amount of peptide-phospholipid complex was isolated. ApoLP-C-III-B showed conformational changes in the circular dichroic spectrum and a shift in the intrinsic tryptophan fluorescence spectrum. Ultracentrifugation in cesium chloride gradients yielded peptide-phospholipid complexes isolated between density 1.10 and 1.18. The molar ratio of lipid to protein was 12:1. The results of these studies and the examination of space filling models of apoLP-C-III provide evidence that an amphipathic alpha helix which contains a nonpolar face and a polar face is the basic structural unit for binding of phospholipid by the plasma apolipoproteins. These results also provide direct evidence that the hydrophobicity of the nonpolar face is important in lipid binding since the nonpolar face of residues 1-40 is considerably less hydrophobic than the nonpolar face of residues 41-79.     

Structural differences in the DNA binding domains of human p53 and its C. elegans ortholog Cep-1

The DNA binding domains of human p53 and Cep-1, its C. elegans ortholog, recognize essentially identical DNA sequences despite poor sequence similarity. We solved the three-dimensional structure of the Cep-1 DNA binding domain in the absence of DNA and compared it to that of human p53. The two domains have similar overall folds. However, three loops, involved in DNA and Zn binding in human p53, contain small alpha helices in Cep-1. The alpha helix in loop L3 of Cep-1 orients the side chains of two conserved arginines toward DNA; in human p53, both arginines are mutation hotspots, but only one contacts DNA. The alpha helix in loop L1 of Cep-1 repositions the entire loop, making it unlikely for residues of this loop to contact bases in the major groove of DNA, as occurs in human p53. Thus, during evolution there have been considerable changes in the structure of the p53 DNA binding domain.     

NMR structure of the human doppel protein

The NMR structure of the recombinant human doppel protein, hDpl(24-152), contains a flexibly disordered "tail" comprising residues 24-51, and a globular domain extending from residues 52 to 149 for which a detailed structure was obtained. The globular domain contains four alpha-helices comprising residues 72-80 (alpha1), 101-115 (alpha2(a)), 117-121 (alpha2(b)), and 127-141 (alpha3), and a short two-stranded anti-parallel beta-sheet comprising residues 58-60 (beta1) and 88-90 (beta2). The fold of the hDpl globular domain thus coincides nearly identically with the structure of the murine Dpl protein. There are close similarities with the human prion protein (hPrP) but, similar to the situation with the corresponding murine proteins, hDpl shows marked local differences when compared to hPrP: the beta-sheet is flipped by 180 degrees with respect to the molecular scaffold formed by the four helices, and the beta1-strand is shifted by two residues toward the C terminus. A large solvent-accessible hydrophobic cleft is formed on the protein surface between beta2 and alpha3, which has no counterpart in hPrP. The helix alpha2 of hPrP is replaced by two shorter helices, alpha2(a) and alpha2(b). The helix alpha3 is shortened by more than two turns when compared with alpha3 of hPrP, which is enforced by the positioning of the second disulfide bond in hDpl. The C-terminal peptide segment 144-149 folds back onto the loop connecting beta2 and alpha2. All but four of the 20 conserved residues in the globular domains of hPrP and hDpl appear to have a structural role in maintaining a PrP-type fold. The conservation of R76, E96, N110 and R134 in hDpl, corresponding to R148, E168, N183 and R208 in hPrP suggests that these amino acid residues might have essential roles in the so far unknown functions of PrP and Dpl in healthy organisms.     

Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.