Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41.

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27-149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30-80) and C-terminal (residues 107-147) helices connected by a 26 residue loop (residues 81-106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N- and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date.     

Biophysical characterization of gp41 aggregates suggests a model for the molecular mechanism of HIV-associated neurological damage and dementia

In human immunodeficiency virus (HIV)-infected individuals, the level of the HIV envelope protein gp41 in brain tissue is correlated with neurological damage and dementia. In this paper we show by biochemical methods and electron microscopy that the extracellular ectodomain of purified HIV and simian immunodeficiency virus gp41 (e-gp41) forms a mixture of soluble high molecular weight aggregate and native trimer at physiological pH. The e-gp41 aggregate is shown to be largely alpha-helical and relatively stable to denaturants. The high molecular weight form of e-gp41 is variable in size ranging from 7 to 70 trimers, which associate by interactions at the interior of the aggregate involving the loop that connects the N- and C-terminal helices of the e-gp41 core. The trimers are predominantly arranged with their long axes oriented radially, and the width of the high molecular weight aggregate corresponds to the length of two e-gp41 trimers (approximately 200 A). Using both light and electron microscopy combined with immunohistochemistry we show that HIV gp41 accumulates as an extracellular aggregate in the brains of HIV-infected patients diagnosed with dementia. We postulate that the high molecular weight aggregates of e-gp41 are responsible for HIV-associated neurological damage and dementia, consistent with known mechanisms of encephalopathy.     

Amino acid sequence analysis of the annexin super-gene family of proteins.

The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried saltbridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family.     

Design, expression, and immunogenicity of a soluble HIV trimeric envelope fragment adopting a prefusion gp41 configuration

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) is comprised of non-covalently associated gp120/gp41 subunits that form trimeric spikes on the virion surface. Upon binding to host cells, Env undergoes a series of structural transitions, leading to gp41 rearrangement necessary for fusion of viral and host membranes. Until now, the prefusion state of gp41 ectodomain (e-gp41) has eluded molecular and structural analysis, and thus assessment of the potential of such an e-gp41 conformer to elicit neutralizing antibodies has not been possible. Considering the importance of gp120 amino (C1) and carboxyl (C5) segments in the association with e-gp41, we hypothesize that these regions are sufficient to maintain e-gp41 in a prefusion state. Based on the available gp120 atomic structure, we designed several truncated gp140 variants by including the C1 and C5 regions of gp120 in a gp41 ectodomain fragment. After iterative cycles of protein design, expression and characterization, we obtained a variant truncated at Lys(665) that stably folds as an elongated trimer under physiologic conditions. Several independent biochemical/biophysical analyses strongly suggest that this mini-Env adopts a prefusion e-gp41 configuration that is strikingly distinct from the postfusion trimer-of-hairpin structure. Interestingly, this prefusion mini-Env, lacking the fragment containing the 2F5/4E10 neutralizing monoclonal antibody binding sites, displays no detectable HIV-neutralizing epitopes when employed as an immunogen in rabbits. The result of this immunogenicity study has important implications for HIV-1 vaccine design efforts. Moreover, this engineered mini-Env protein should facilitate three-dimensional structural studies of the prefusion e-gp41 and serve to guide future attempts at pharmacologic and immunologic intervention of HIV-1.     

Structural and functional analysis of interhelical interactions in the human immunodeficiency virus type 1 gp41 envelope glycoprotein by alanine-scanning mutagenesis

Membrane fusion by human immunodeficiency virus type 1 (HIV-1) is promoted by the refolding of the viral envelope glycoprotein into a fusion-active conformation. The structure of the gp41 ectodomain core in its fusion-active state is a trimer of hairpins in which three antiparallel carboxyl-terminal helices pack into hydrophobic grooves on the surface of an amino-terminal trimeric coiled coil. In an effort to identify amino acid residues in these grooves that are critical for gp41 activation, we have used alanine-scanning mutagenesis to investigate the importance of individual side chains in determining the biophysical properties of the gp41 core and the membrane fusion activity of the gp120-gp41 complex. Alanine substitutions at Leu-556, Leu-565, Val-570, Gly-572, and Arg-579 positions severely impaired membrane fusion activity in envelope glycoproteins that were for the most part normally expressed. Whereas alanine mutations at Leu-565 and Val-570 destabilized the trimer-of-hairpins structure, mutations at Gly-572 and Arg-579 led to the formation of a stable gp41 core. Our results suggest that the Leu-565 and Val-570 residues are important determinants of conserved packing interactions between the amino- and carboxyl-terminal helices of gp41. We propose that the high degree of sequence conservation at Gly-572 and Arg-579 may result from selective pressures imposed by prefusogenic conformations of the HIV-1 envelope glycoprotein. Further analysis of the gp41 activation process may elucidate targets for antiviral intervention.     

Monomer-trimer equilibrium of the ectodomain of SIV gp41: insight into the mechanism of peptide inhibition of HIV infection.

The monomer-trimer equilibrium of the ectodomain of SIV gp41 (residues 27-149, e-gp41) has been characterized by analytical ultracentrifugation, circular dichroism (CD), and NMR spectroscopy. Based on analytical ultracentrifugation experiments performed at different rotor speeds and protein concentrations, the equilibrium association constant for the SIV e-gp41 trimer is 3.1 x 10(11) M(-2). The presence of intermolecular nuclear Overhauser effects in a mixture of 12C and 13C-labeled e-gp41 prepared under nondenaturing conditions unambiguously demonstrates that there is a dynamic equilibrium between the monomer and trimer. The CD spectra taken as a function of SIV e-gp41 concentration suggest that the helical content of the monomeric state does not change significantly relative to that of the trimeric state. The relevance of the monomer-trimer equilibrium is discussed with respect to gp41 function and the inhibitory properties of gp41 peptides.     

Heterogeneous patterns of variation among multiple human x-linked Loci: the possible role of diversity-reducing selection in non-africans

Studies of human DNA sequence polymorphism reveal a range of diversity patterns throughout the genome. This variation among loci may be due to natural selection, demographic influences, and/or different sampling strategies. Here we build on a continuing study of noncoding regions on the X chromosome in a panel of 41 globally sampled humans representing African and non-African populations by examining patterns of DNA sequence variation at four loci (APXL, AMELX, TNFSF5, and RRM2P4) and comparing these patterns with those previously reported at six loci in the same panel of 41 individuals. We also include comparisons with patterns of noncoding variation seen at five additional X-linked loci that were sequenced in similar global panels. We find that, while almost all loci show a reduction in non-African diversity, the magnitude of the reduction varies substantially across loci. The large observed variance in non-African levels of diversity results in the rejection of a neutral model of molecular evolution with a multi-locus HKA test under both a constant size and a bottleneck model. In non-Africans, some loci harbor an excess of rare mutations over neutral equilibrium predictions, while other loci show no such deviation in the distribution of mutation frequencies. We also observe a positive relationship between recombination rate and frequency spectra in our non-African, but not in our African, sample. These results indicate that a simple out-of-Africa bottleneck model is not sufficient to explain the observed patterns of sequence variation and that diversity-reducing selection acting at a subset of loci and/or a more complex neutral model must be invoked.     

mRNA sequence predictions from homologous protein sequences

A model has been developed that permits the prediction of mRNA nucleic acid sequence from the sequences of the translated proteins. The model relies on the information obtained from the comparison of protein sequences in related species to reduce the number of possible codons for those amino acids where mutations are observed. The predictions so obtained have been tested by applying the model to proteins whose mRNA sequences are known. The model's predictions have been found to be 100% accurate if three or more different amino acids are known at a given position and if the protein sequences are restricted to relatively closely related species (within the same class). The use of this model may permit a reduction of the mRNA sequence degeneracy and therefore be helpful in the synthesis of cDNA probes or for the prediction of restriction endonuclease sites. Computer programs have been developed to ease the use of the model.     

A Calpha model for the transmembrane alpha helices of gap junction intercellular channels

Gap junction channels connect the cytoplasms of apposed cells via an intercellular conduit formed by the end-to-end docking of two hexameric hemichannels called connexons. We used electron cryomicroscopy to derive a three-dimensional density map at 5.7 angstroms in-plane and 19.8 angstroms vertical resolution, allowing us to identify the positions and tilt angles for the 24 alpha helices within each hemichannel. The four hydrophobic segments in connexin sequences were assigned to the alpha helices in the map based on biochemical and phylogenetic data. Analyses of evolutionary conservation and compensatory mutations in connexin evolution identified the packing interfaces between the helices. The final model, which specifies the coordinates of Calpha atoms in the transmembrane domain, provides a structural basis for understanding the different physiological effects of almost 30 mutations and polymorphisms in terms of structural deformations at the interfaces between helices, revealing an intimate connection between molecular structure and disease.     

Interhelical packing modulates conformational flexibility in the lactose permease of Escherichia coli

A key to obtaining an X-ray structure of the lactose permease of Escherichia coli (LacY) (Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., and Iwata, S. (2003) Science 301, 549-716) was the use of a mutant in which Cys154 (helix V) is replaced with Gly. LacY containing this mutation strongly favors an inward-facing conformation, which binds ligand with high affinity, but catalyzes little transport and exhibits few if any of the ligand-dependent conformational changes observed with wild-type LacY. The X-ray structure demonstrates that helix V crosses helix I in the approximate middle of the membrane in such a manner that Cys154 lies close to Gly24 (helix I). Therefore, it seems likely that replacing Cys154 with Gly may lead to tighter packing between helices I and V, thereby resulting in the phenotype observed. Consistently, replacement of Gly24 with Cys in the C154G mutant rescues significant transport activity, and the mutant exhibits properties similar to wild-type LacY with respect to substrate binding and thermostability. However, the only other replacements that rescue transport to any extent whatsoever are Val and Asp, both of which are much less effective than Cys. The results suggest that, although helix packing probably plays an important role with respect to the properties of the C154G mutant, the ability of Cys at position 24 to rescue transport activity of C154G is more complicated than simple replacement of bulk between positions 24 and 154. Rather, activity is dependent on more subtle interactions between the helices, and mutations that disrupt interactions between helix IV and loop 6-7 or between helices II and IV also rescue transport in the C154G mutant.     

Mutational Analysis of Residues in the Coiled-Coil Domain of Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41

The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263–273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426–430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.     

Conservation Weighting Functions Enable Covariance Analyses to Detect Functionally Important Amino Acids

The explosive growth in the number of protein sequences gives rise to the possibility of using the natural variation in sequences of homologous proteins to find residues that control different protein phenotypes. Because in many cases different phenotypes are each controlled by a group of residues, the mutations that separate one version of a phenotype from another will be correlated. Here we incorporate biological knowledge about protein phenotypes and their variability in the sequence alignment of interest into algorithms that detect correlated mutations, improving their ability to detect the residues that control those phenotypes. We demonstrate the power of this approach using simulations and recent experimental data. Applying these principles to the protein families encoded by Dscam and Protocadherin allows us to make testable predictions about the residues that dictate the specificity of molecular interactions.     

Transmembrane structure of an inwardly rectifying potassium channel

Inwardly rectifying potassium channels (K(ir)), comprising four subunits each with two transmembrane domains, M1 and M2, regulate many important physiological processes. We employed a yeast genetic screen to identify functional channels from libraries of K(ir) 2.1 containing mutagenized M1 or M2 domains. Patterns in the allowed sequences indicate that M1 and M2 are helices. Protein-lipid and protein-water interaction surfaces identified by the patterns were verified by sequence minimization experiments. Second-site suppressor analyses of helix packing indicate that the M2 pore-lining inner helices are surrounded by the M1 lipid-facing outer helices, arranged such that the M1 helices participate in subunit-subunit interactions. This arrangement is distinctly different from the structure of a bacterial potassium channel with the same topology and identifies helix-packing residues as hallmark sequences common to all K(ir) superfamily members.     

Genetic diversity of nuclear ITS1-5.8S-ITS2 rDNA sequence in Clonorchis sinensis Cobbold, 1875 (Trematoda: Opisthorchidae) from the Russian Far East

The present study examined the molecular organisation and sequence variation in the nuclear ribosomal DNA (rDNA) region, including the two internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the Clonorchis sinensis from the Russian Far East. The relevant sequences from other parts of this species' area were downloaded from GenBank. The results showed 100% identity for all investigated 5.8S-ITS2 rDNA sequences. In contrast, two levels of intraspecific variations were revealed in the complete ITS1 sequences. The intra-genomic variation resulted from a C/T polymorphism in a single position. The inter-individual differences between the ITS1 sequences were both due to nucleotide and size polymorphisms resulting from a varying number of five-nucleotide repeats and followed by two ITS1 length variants. These variant frequencies correlate with the clonorchiasis level in some geographical localities. ITS1 differences, both in the mutation profile and mutation localisation, were revealed between northern and southern geographical samples. The presence of GC boxes that are identical to known regulatory motifs in eukaryotes was detected within the ITS1 sub-repeats. The predicted secondary structures for ITS1 consist of two large branches, one of which was invariable, while another depended on ITS1 length. The predicted secondary structure for ITS2 includes four helices around the core. The main differences between C. sinensis and other opisthorchids were localised on the tops of helices 2, 3, and 4. A phylogenetic MST reconstruction subdivided all ITS1 sequences into two well differentiated clusters, each with the major widespread ribotype, and showed that ribotype diversity in both Russia and Korea is much lower than in China. The results obtained demonstrate the feasibility of complete ITS1 sequences in C. sinensis population genetics and can be considered as a basis for further studies of the parasite infection because they may help to elucidate the molecular mechanisms of pathogen evolution and adaptation.     

Packing of α-helices: Geometrical constraints and contact areas

The helical packing in sperm whale myoglobin has been examined. Using cylindrical co-ordinates based on each helix axis in turn, the overlap of the side-chain atoms of a helix with the surrounding atoms from other parts of the structure was 2.3 Å, but the distribution was not at all uniform and severe overlap occurred in at least one location for each helix. Simple axial translations or rotations of any helix in the native structure are not permitted motions. Translation perpendicular to the helix axis in at least one direction is not restricted by interlocking side-chains.The approach of two helices along the contact normal connecting their axes produces solvent exclusion effects at a distance of about 6 Å from the final position. The solvent-excluded area found in such interaction sites is equivalent to a large hydrophobic contribution to the free energy of association. The six principal sites correspond by themselves to 40% of the total area change in going from the extended sausage model to the native structure. The mean atom-packing densities for these sites and the standard deviations of these values are similar and are equal to that found for the protein as a whole.Helices of close-packed spheres form useful approximations to actual peptide helices. The helix of index number four corresponds closely to an α-helix. The required sphere size corresponds in volume to residues such as leucine or methionine. The predicted packing scheme for such helices corresponds to the three general classes of interactions actually seen.Making use of the geometry implied by the close-packed sphere helix, an algorithm is proposed for picking potentially strong helix-helix interaction sites in peptide chains of known sequence. When combined with preliminary secondary structure predictions, it is suggested that this algorithm might usefully restrict the search for these specific types of contact in the docking portion of a general folding program.     

Effect of sequence hydrophobicity and bilayer width upon the minimum length required for the formation of transmembrane helices in membranes

The minimum hydrophobic length necessary to form a transmembrane (TM) helix in membranes was investigated using model membrane-inserted hydrophobic helices. The fluorescence of a Trp at the center of the sequence and its sensitivity to quenching were used to ascertain helix position within the membrane. Peptides with hydrophobic cores composed of poly(Leu) were compared to sequences containing a poly 1:1 Leu:Ala core (which have a hydrophobicity typical of natural TM helices). Studies varying bilayer width revealed that the poly(Leu) core peptides predominately formed a TM state when the bilayer width exceeded hydrophobic sequence length by (i.e. when negative mismatch was) up to ∼ 11-12 Å (e.g. the case of a 11-12 residue hydrophobic sequence in bilayers with a biologically relevant width, i.e. dioleoylphosphatidylcholine (DOPC) bilayers), while poly(LeuAla) core peptides formed predominantly TM state with negative mismatch of up to 9 Å (a 13 residue hydrophobic sequence in DOPC bilayers). This indicates that minimum length necessary to form a predominating amount of a TM state (minimum TM length) is only modestly hydrophobicity-dependent for the sequences studied here, and a formula that defines the minimum TM length as a function of hydrophobicity for moderately-to-highly hydrophobic sequences was derived. The minimum length able to form a stable TM helix for alternating LeuAla sequences, and that for sequences with a Leu block followed by an Ala block, was similar, suggesting that a hydrophobicity gradient along the sequence may not be an important factor in TM stability. TM stability was also similar for sequences flanked by different charged ionizable residues (Lys, His, Asp). However, ionizable flanking residues destabilized the TM configuration much more when charged than when uncharged. The ability of short hydrophobic sequences to form TM helices in membranes in the presence of substantial negative mismatch implies that lipid bilayers have a considerable ability to adjust to negative mismatch, and that short TM helices may be more common than generally believed. Factors that modulate the ability of bilayers to adjust to mismatch may strongly affect the configuration of short hydrophobic helices.     

General architecture of the alpha-helical globule

A model is presented for the arrangement of alpha-helices in globular proteins. In the model, helices are placed on certain ribs of "quasi-spherical" polyhedra. The polyhedra are chosen so as to allow the close packing of helices around a hydrophobic core and to stress the collective interactions of the individual helices. The model predicts a small set of stable architectures for alpha-helices in globular proteins and describes the geometries of the helix packings. Some of the predicted helix arrangements have already been observed in known protein structures; others are new. An analysis of the three-dimensional structures of all proteins for which co-ordinates are available shows that the model closely approximates the arrangements and packing of helices actually observed. The average deviations of the real helix axes from those in the model polyhedra is +/- 20 degrees in orientation and +/- 2 A in position (1 A = 0.1 nm). We also show that for proteins that are not homologous, but whose helix arrangements are described by the same polyhedron, the root-mean-square difference in the position of the C alpha atoms in the helices is 1.6 to 3.0 A.     

Packing of transmembrane helices in bacteriorhodopsin folding: structure and thermodynamics

We propose a coarse-grained (CG) model to study the native structure and physical properties of helical membrane proteins (HMPs) using off-lattice computer simulations. Instead of considering sequence heterogeneity explicitly, we model its effect on the packing of helices by employing a mean packing parameter r(0), which is calculated from an all-atom (AA) model. Specifically, this CG model is applied to investigate the packing of helices in bacteriorhodopsin (BR), and predicts the seven helix bundle structure of BR with a root mean square deviation (RMSD) in coordinates of helix backbone atoms (N, C, C(alpha)) of 3.99 A from its crystal structure. This predicted structure is further refined in an AA model by Amber and the refined structure has a RMSD (in coordinates of helix backbone atoms) of 2.64 A. The predicted packing position, tilting angle, and orientation angle of each helix in the refined structure are consistent with experimental data and their physical origins can be well understood in our model. Our results show that a reasonably good structure of BR can be predicted by using such a dual-scale approach, provided that its secondary structure is known. Starting from a random initial configuration, the folded structure can be obtained in days using a regular desktop computer. Various thermodynamic properties of helix packing of BR are also investigated in this CG model.     

Crystal structures of disordered Z-type helices

The X-ray crystal structures of a decamer sequence d(CGCGTACGCG)₂ and a tetradecamer sequence d(CGCGCGTACGCGCG)₂ are presented here. Both sequences are alternating pyrimidine-purine repeat sequences and they form disordered, pseudo-continuous left handed Z-type helices. They demonstrate interesting variants of the ‘bundles of columns of helices’ mode of packing.