ABSTRACT
A simple NMR experiment for the measurement of transverse relaxation rates of degenerate 1H spins in 13CH3 methyl groups of deuterated proteins, is described. The experiment relies on the use of acute-angle 1H radio-frequency pulses, whereby a series of methyl 1H R2 decays is acquired with different values of the 1H pulse flip-angle, which modulates the relative contributions of the slow- and fast-relaxing components of 1H magnetization during the relaxation delay. These are subsequently analyzed simultaneously to extract the transverse relaxation rates of both components (RS and RF), along with the rate of cross-relaxation between these components, δ. The dipolar 1H-1H cross-correlated relaxation rate η = (RF - RS)/2 and the cross-relaxation rate δ, extracted from such acute-angle-pulse R2 decay series are compared with those derived from well-established methodology that uses relaxation-violated methyl 1H coherence transfer (so-called 'forbidden' experiments). Good agreement is achieved for the rates η derived from the two techniques, while slightly more accurate values of δ are obtained from analysis of acute-angle-pulse R2 series. Recording of acute-angle-pulse R2 series represents an attractive alternative to existing methodologies for quantifying methyl 1H relaxation and dynamics of the methyl three-fold symmetry axis in selectively [13CH3]-methyl-labeled, deuterated proteins - particularly so for very high-molecular-weight proteins, where the measurements of RF rates or the build-up of methyl 1H magnetization in relaxation-violated coherence transfer experiments, are problematic due to low sensitivity.
Subject(s)
Magnetic Resonance Imaging , Proteins , Magnetic Resonance Spectroscopy , Nuclear Magnetic Resonance, BiomolecularABSTRACT
We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E. coli BL21(DE3) cells in shaker flasks that reduces D2O usage tenfold and d7-glucose usage by 30 %. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD600 of up to 10. Inducing the cells with isopropyl ß-D-1-thiogalactopyranoside at an OD600 of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD600 between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of E. coli from H2O-based to D2O-based medium is also key for ensuring high levels of protein expression in D2O. We find that a simple three-step adaptation approach-Luria-Bertani (LB) medium in H2O to LB in D2O to modified-M9 medium in D2O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.
Subject(s)
Batch Cell Culture Techniques , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
We present a software module which allows one to efficiently perform molecular dynamics and local minimization calculations in internal coordinates when incorporated into a molecular dynamics package. We have implemented a reference interface to the NIH version of the X-PLOR structure refinement package and we show that the module provides superior torsion-angle dynamics functionality relative to the native X-PLOR implementation. The module has been designed in a portable fashion so that interfacing it with other packages should be relatively easy. Other features of the module include the ability to define rather general internal coordinates, an accurate integration algorithm which can automatically adjust the integration step size, and a modular design, which facilitates extending and enhancing the module.
Subject(s)
Bacterial Proteins , Magnetic Resonance Spectroscopy , Nerve Tissue Proteins/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Software , Algorithms , Protein ConformationABSTRACT
The HMG-box domain of the human male sex-determining factor SRY, hSRY(HMG) (comprising residues 57-140 of the full-length sequence), binds DNA sequence-specifically in the minor groove, resulting in substantial DNA bending. The majority of point mutations resulting in 46X,Y sex reversal are located within this domain. One clinical de novo mutation, M64I in the full-length hSRY sequence, which corresponds to M9I in the present hSRY(HMG) construct, acts principally by reducing the extent of DNA bending. To elucidate the structural consequences of the M9I mutation, we have solved the 3D solution structures of wild-type and M9I hSRY(HMG) complexed to a DNA 14mer by NMR, including the use of residual dipolar couplings to derive long-range orientational information. We show that the average bend angle (derived from an ensemble of 400 simulated annealing structures for each complex) is reduced by approximately 13 degrees from 54(+/-2) degrees in the wild-type complex to 41(+/-2) degrees in the M9I complex. The difference in DNA bending can be localized directly to changes in roll and tilt angles in the ApA base-pair step involved in interactions with residue 9 and partial intercalation of Ile13. The larger bend angle in the wild-type complex arises as a direct consequence of steric repulsion of the sugar of the second adenine by the bulky S(delta) atom of Met9, whose position is fixed by a hydrogen bond with the guanidino group of Arg17. In the M9I mutant, this hydrogen bond can no longer occur, and the less bulky C(gamma)m methyl group of Ile9 braces the sugar moieties of the two adenine residues, thereby decreasing the roll and tilt angles at the ApA step by approximately 8 degrees and approximately 5 degrees, respectively, and resulting in an overall difference in bend angle of approximately 13 degrees between the two complexes. To our knowledge, this is one of the first examples where the effects of a clinical mutation involving a protein-DNA complex have been visualized at the atomic level.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , Gonadal Dysgenesis, 46,XY/genetics , Nuclear Proteins , Nucleic Acid Conformation , Point Mutation/genetics , Transcription Factors , Amino Acid Motifs , Binding Sites , DNA/genetics , DNA-Binding Proteins/genetics , Disorders of Sex Development , Female , High Mobility Group Proteins/chemistry , Humans , Hydrogen Bonding , Male , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Tertiary , Sex Determination Processes , Sex-Determining Region Y Protein , SolutionsABSTRACT
The nuclear envelope proteins LAP2, emerin and MAN1 share a conserved approximately 40-residue 'LEM' motif. Loss of emerin causes Emery-Dreifuss muscular dystrophy. We have solved the solution NMR structure of the constant region of human LAP2 (residues 1-168). Human LAP2(1-168) has two structurally independent, non-interacting domains located at residues 1-50 ('LAP2-N') and residues 111-152 (LEM-domain), connected by an approximately 60-residue flexible linker. The two domains are structurally homologous, comprising a helical turn followed by two helices connected by an 11-12-residue loop. This motif is shared by subdomains of T4 endonuclease VII and transcription factor rho, despite negligible (< or =15%) sequence identity. NMR chemical shift mapping demonstrated that the LEM-domain binds BAF (barrier-to-autointegration factor), whereas LAP2-N binds DNA. Both binding surfaces comprise helix 1, the N-terminus of helix 2 and the inter-helical loop. Binding selectivity is determined by the nature of the surface residues in these binding sites, which are predominantly positively charged for LAP2-N and hydrophobic for the LEM-domain. Thus, LEM and LEM-like motifs form a common structure that evolution has customized for binding to BAF or DNA.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , Membrane Proteins/chemistry , Nuclear Proteins , Amino Acid Sequence , DNA/chemistry , Humans , Membrane Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Nuclear Envelope/metabolism , Nucleoproteins/metabolism , Protein Structure, Tertiary , SolutionsABSTRACT
NMR structure determination of nucleic acids presents an intrinsically difficult problem since the density of short interproton distance contacts is relatively low and limited to adjacent base pairs. Although residual dipolar couplings provide orientational information that is clearly helpful, they do not provide translational information of either a short-range (with the exception of proton-proton dipolar couplings) or long-range nature. As a consequence, the description of the nonbonded contacts has a major impact on the structures of nucleic acids generated from NMR data. In this paper, we describe the derivation of a potential of mean force derived from all high-resolution (2 A or better) DNA crystal structures available in the Nucleic Acid Database (NDB) as of May 2000 that provides a statistical description, in simple geometric terms, of the relative positions of pairs of neighboring bases (both intra- and interstrand) in Cartesian space. The purpose of this pseudopotential, which we term a DELPHIC base-base positioning potential, is to bias sampling during simulated annealing refinement to physically reasonable regions of conformational space within the range of possibilities that are consistent with the experimental NMR restraints. We illustrate the application of the DELPHIC base-base positioning potential to the structure refinement of a DNA dodecamer, d(CGCGAATTCGCG)(2), for which NOE and dipolar coupling data have been measured in solution and for which crystal structures have been determined. We demonstrate by cross-validation against independent NMR observables (that is, both residual dipolar couplings and NOE-derived intereproton distance restraints) that the DELPHIC base-base positioning potential results in a significant increase in accuracy and obviates artifactual distortions in the structures arising from the limitations of conventional descriptions of the nonbonded contacts in terms of either Lennard-Jones van der Waals and electrostatic potentials or a simple van der Waals repulsion potential. We also demonstrate, using experimental NMR data for a complex of the male sex determining factor SRY with a duplex DNA 14mer, which includes a region of highly unusual and distorted DNA, that the DELPHIC base-base positioning potential does not in any way hinder unusual interactions and conformations from being satisfactorily sampled and reproduced. We expect that the methodology described in this paper for DNA can be equally applied to RNA, as well as side chain-side chain interactions in proteins and protein-protein complexes, and side chain-nucleic acid interactions in protein-nucleic acid complexes. Further, this approach should be useful not only for NMR structure determination but also for refinement of low-resolution (3-3.5 A) X-ray data.
Subject(s)
DNA/chemistry , Magnetic Resonance Spectroscopy/methods , Databases as Topic , Models, Molecular , Molecular StructureABSTRACT
The design and characterization of a chimeric protein, termed N(CCG)-gp41, derived from the ectodomain of human immunodeficiency virus (HIV), type I gp41 is described. N(CCG)-gp41 features an exposed trimeric coiled-coil comprising the N-terminal helices of the gp41 ectodomain. The trimeric coiled-coil is stabilized both by fusion to a minimal thermostable ectodomain of gp41 and by engineered intersubunit disulfide bonds. N(CCG)-gp41 is shown to inhibit HIV envelope-mediated cell fusion at nanomolar concentrations with an IC(50) of 16.1 +/- 2.8 nm. It is proposed that N(CCG)-gp41 targets the exposed C-terminal region of the gp41 ectodomain in its pre-hairpin intermediate state, thereby preventing the formation of the fusogenic form of the gp41 ectodomain, which comprises a highly stable trimer of hairpins arranged in a six-helix bundle. N(CCG)-gp41 has potential as a therapeutic agent for the direct inhibition of HIV cell entry, as an anti-HIV vaccine, and as a component of a rapid throughput assay for screening for small molecule inhibitors of HIV envelope-mediated cell fusion. It is anticipated that antibodies raised against N(CCG)-gp41 may target the trimeric coiled-coil of N-terminal helices of the gp41 ectodomain that is exposed in the pre-hairpin intermediate state in a manner analogous to peptides derived from the C-terminal helix of gp41 that are currently in clinical trials.
Subject(s)
Anti-HIV Agents/chemistry , Cell Fusion , HIV Envelope Protein gp41/biosynthesis , HIV Envelope Protein gp41/genetics , HIV-1/physiology , Recombinant Fusion Proteins/biosynthesis , 3T3 Cells , AIDS Vaccines/biosynthesis , AIDS Vaccines/chemistry , Amino Acid Sequence , Animals , Anti-HIV Agents/pharmacology , Base Sequence , CD4 Antigens/physiology , Cell Line , Circular Dichroism , Crystallography, X-Ray , Disulfides , Drug Design , HIV Envelope Protein gp41/chemistry , HIV-1/drug effects , HIV-1/genetics , Humans , Mice , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Engineering , Protein Folding , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacology , Spectrophotometry, Ultraviolet , Viral Envelope Proteins/physiologyABSTRACT
The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) includes a collection of proteins that accomplish phosphoryl transfer from phosphoenolpyruvate (PEP) to a sugar in the course of transport. The soluble proteins of the glucose transport pathway also function as regulators of diverse systems. The mechanism of interaction of the phosphoryl carrier proteins with each other as well as with their regulation targets has been amenable to study by nuclear magnetic resonance (NMR) spectroscopy. The three-dimensional solution structures of the complexes between the N-terminal domain of enzyme I and HPr and between HPr and enzyme IIA(Glc) have been elucidated. An analysis of the binding interfaces of HPr with enzyme I, IIA(Glc) and glycogen phosphorylase revealed that a common surface on HPr is involved in all these interactions. Similarly, a common surface on IIA(Glc) interacts with HPr, IIB(Glc) and glycerol kinase. Thus, there is a common motif for the protein-protein interactions characteristic of the PTS.
Subject(s)
Escherichia coli/enzymology , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Binding Sites , Escherichia coli Proteins , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Phosphoenolpyruvate/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Protein Conformation , Protein Structure, SecondaryABSTRACT
In this paper we present the VMD-XPLOR package combining the XPLOR refinement program and the VMD visualization program and including extensions for use in the determination of biomolecular structures from NMR data. The package allows one to pass structures to and to control VMD from the XPLOR scripting level. The VMD graphical interface has been customized for NMR structure refinement, including support to manipulate coordinates interactively while graphically visualizing NMR experimental information in the context of a molecular structure. Finally, the VMD-XPLOR interface is modular so that it is readily transferable to other refinement programs (such as CNS).
Subject(s)
Bacterial Proteins , Computer Graphics , Nuclear Magnetic Resonance, Biomolecular/methods , Software , Molecular Structure , Nerve Tissue Proteins/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Protein ConformationABSTRACT
Molecular replacement using search models derived from nuclear magnetic resonance (NMR) spectroscopy has often proved problematic. It has been known for some time that the overall differences in atomic positions (r.m.s.d.) between the crystalline and the solution states of the same protein are of the order of 1-2 A and approach the limit of molecular replacement. In most cases, this structural difference is a result of calculating the NMR structure with insufficient data, yielding an NMR structure of limited accuracy. A systematic case study was performed to investigate the use of NMR models for molecular replacement on the p53 tetramerization domain: NMR search models of varying degrees of accuracy were employed to solve phases for the 1.5 A X-ray diffraction data. An approximate correlation was found between the accuracy of the NMR search model and the clarity and quality of the molecular-replacement solution. It was found that ensemble models perform better than single averaged models and have a larger tolerance in model inaccuracy. Also, distance-derived B factors can improve the performance of single models.
Subject(s)
Magnetic Resonance Spectroscopy/methods , Tumor Suppressor Protein p53/chemistry , Crystallography, X-Ray/methods , Humans , Models, Chemical , Protein ConformationABSTRACT
The solution structure of the second protein-protein complex of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system, that between histidine-containing phosphocarrier protein (HPr) and glucose-specific enzyme IIA(Glucose) (IIA(Glc)), has been determined by NMR spectroscopy, including the use of dipolar couplings to provide long-range orientational information and newly developed rigid body minimization and constrained/restrained simulated annealing methods. A protruding convex surface on HPr interacts with a complementary concave depression on IIA(Glc). Both binding surfaces comprise a central hydrophobic core region surrounded by a ring of polar and charged residues, positive for HPr and negative for IIA(Glc). Formation of the unphosphorylated complex, as well as the phosphorylated transition state, involves little or no change in the protein backbones, but there are conformational rearrangements of the interfacial side chains. Both HPr and IIA(Glc) recognize a variety of structurally diverse proteins. Comparisons with the structures of the enzyme I-HPr and IIA(Glc)-glycerol kinase complexes reveal how similar binding surfaces can be formed with underlying backbone scaffolds that are structurally dissimilar and highlight the role of redundancy and side chain conformational plasticity.
Subject(s)
Bacterial Proteins/chemistry , Escherichia coli/enzymology , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Binding Sites , Escherichia coli/genetics , Escherichia coli Proteins , Glycerol Kinase/chemistry , Glycerol Kinase/genetics , Macromolecular Substances , Magnetic Resonance Spectroscopy , Models, Molecular , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Protein Conformation , Signal Transduction , SolutionsABSTRACT
Enzyme IIA(Glucose) (IIA(Glc)) is a signal-transducing protein in the phosphotransferase system of Escherichia coli. Structural studies of free IIA(Glc) and the HPr-IIA(Glc) complex have shown that IIA(Glc) comprises a globular beta-sheet sandwich core (residues 19-168) and a disordered N-terminal tail (residues 1-18). Although the presence of the N-terminal tail is not required for IIA(Glc) to accept a phosphorus from the histidine phosphocarrier protein HPr, its presence is essential for effective phosphotransfer from IIA(Glc) to the membrane-bound IIBC(Glc). The sequence of the N-terminal tail suggests that it has the potential to form an amphipathic helix. Using CD, we demonstrate that a peptide, corresponding to the N-terminal 18 residues of IIA(Glc), adopts a helical conformation in the presence of either the anionic lipid phosphatidylglycerol or a mixture of anionic E. coli lipids phosphatidylglycerol (25%) and phosphatidylethanolamine (75%). The peptide, however, is in a random coil state in the presence of the zwitterionic lipid phosphatidylcholine, indicating that electrostatic interactions play a role in the binding of the lipid to the peptide. In addition, we show that intact IIA(Glc) also interacts with anionic lipids, resulting in an increase in helicity, which can be directly attributed to the N-terminal segment. From these data we propose that IIA(Glc) comprises two functional domains: a folded domain containing the active site and capable of weakly interacting with the peripheral IIB domain of the membrane protein IIBC(Glc); and the N-terminal tail, which interacts with the negatively charged E. coli membrane, thereby stabilizing the complex of IIA(Glc) with IIBC(Glc). This stabilization is essential for the final step of the phosphoryl transfer cascade in the glucose transport pathway.
Subject(s)
Escherichia coli/enzymology , Membrane Proteins/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Signal Transduction , Amino Acid Sequence , Escherichia coli Proteins , Membrane Proteins/chemistry , Molecular Sequence Data , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistryABSTRACT
It is often the case that a substantial number of torsion angles (both backbone and sidechain) in structures of proteins and nucleic acids determined by NMR are found in physically unlikely and energetically unfavorable conformations. We have previously proposed a database-derived potential of mean force comprising one-, two-, three-, and four-dimensional potential surfaces which describe the likelihood of various torsion angle combinations to bias conformational sampling during simulated annealing refinement toward those regions that are populated in very high resolution (< or =1.75 A) crystal structures. We now note a shortcoming of our original implementation of this approach: namely, the forces it places on atoms are very rough. When the density of experimental restraints is low, this roughness can both hinder convergence to commonly populated regions of torsion angle space and reduce overall conformational sampling. In this paper we describe a modification that completely eliminates these problems by replacing the original potential surfaces by a sum of multidimensional Gaussian functions. Structures refined with the new Gaussian implementation now simultaneously enjoy excellent global sampling and excellent local choices of torsion angles.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Proteins/chemistry , Algorithms , Protein ConformationABSTRACT
A simple and rapid method is presented for solving the three-dimensional structures of protein-protein complexes in solution on the basis of experimental NMR restraints that provide the requisite translational (i.e., intermolecular nuclear Overhauser enhancement, NOE, data) and orientational (i.e., backbone (1)H-(15)N dipolar couplings and intermolecular NOEs) information. Providing high-resolution structures of the proteins in the unbound state are available and no significant backbone conformational changes occur upon complexation (which can readily be assessed by analysis of dipolar couplings measured on the complex), accurate and rapid docking of the two proteins can be achieved. The method, which is demonstrated for the 40-kDa complex of enzyme I and the histidine phosphocarrier protein, involves the application of rigid body minimization using a target function comprising only three terms, namely experimental NOE-derived intermolecular interproton distance and dipolar coupling restraints, and a simple intermolecular van der Waals repulsion potential. This approach promises to dramatically reduce the amount of time and effort required to solve the structures of protein-protein complexes by NMR, and to extend the capabilities of NMR to larger protein-protein complexes, possibly up to molecular masses of 100 kDa or more.
Subject(s)
Proteins/metabolism , Magnetic Resonance Spectroscopy , Protein Binding , Protein Conformation , Proteins/chemistryABSTRACT
The solution structure of the 33 kDa complex between the dimeric DNA-binding core domain of the transcription factor MEF2A (residues 1-85) and a 20mer DNA oligonucleotide comprising the consensus sequence CTA(A/T)(4)TAG has been solved by NMR. The protein comprises two domains: a MADS-box (residues 1-58) and a MEF2S domain (residues 59-73). Recognition and specificity are achieved by interactions between the MADS-box and both the major and minor grooves of the DNA. A number of critical differences in protein-DNA contacts observed in the MEF2A-DNA complex and the DNA complexes of the related MADS-box transcription factors SRF and MCM1 provide a molecular explanation for modulation of sequence specificity and extent of DNA bending ( approximately 15 versus approximately 70 degrees ). The structure of the MEF2S domain is entirely different from that of the equivalent SAM domain in SRF and MCM1, accounting for the absence of cross-reactivity with other proteins that interact with these transcription factors.
Subject(s)
DNA-Binding Proteins/chemistry , DNA/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Cross Reactions , DNA/metabolism , DNA-Binding Proteins/metabolism , Dimerization , Humans , MADS Domain Proteins , MEF2 Transcription Factors , Macromolecular Substances , Magnetic Resonance Spectroscopy , Minichromosome Maintenance 1 Protein , Models, Molecular , Molecular Sequence Data , Myogenic Regulatory Factors , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleic Acid Conformation/drug effects , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Sequence Alignment , Sequence Homology, Amino Acid , Serum Response Factor , Solutions , Structure-Activity Relationship , Transcription Factors/classification , Transcription Factors/metabolism , Transcription Factors/pharmacologyABSTRACT
The bacterial phosphoenolpyruvate:sugar phosphotransferase system accomplishes both the transport and phosphorylation of sugars as well as the regulation of some cellular processes. An important component of this system is the histidine-containing phosphocarrier protein, HPr, which accepts a phosphoryl group from enzyme I, transfers a phosphoryl group to IIA proteins, and is an allosteric regulator of glycogen phosphorylase. Because the nature of the surface on HPr that interacts with this multiplicity of proteins from Escherichia coli was previously undefined, we investigated these interactions by nuclear magnetic resonance spectroscopy. The chemical shift changes of the backbone and side-chain amide (1)H and (15)N nuclei of uniformly (15)N-labeled HPr in the absence and presence of natural abundance glycogen phosphorylase, glucose-specific enzyme IIA, or the N-terminal domain of enzyme I have been determined. Mapping these chemical shift perturbations onto the three-dimensional structure of HPr permitted us to identify the binding surface(s) of HPr for interaction with these proteins. Here we show that the mapped interfaces on HPr are remarkably similar, indicating that HPr employs a similar surface in binding to its partners.
Subject(s)
Bacterial Proteins/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Bacterial Proteins/chemistry , Escherichia coli/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Protein BindingABSTRACT
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.