Tobacco mosaic virus protein aggregates in solution: structural comparison of 20S aggregates with those near conditions for disk crystallization

Previous X-ray studies (2.8-A resolution) on the crystals of tobacco mosaic virus protein (TMVP) grown from solutions containing high salt have characterized the structure of the protein aggregate as a bilayered cylindrical disk formed by 34 identical subunits [Bloomer, A.C., Champness, J.N., Bricogne, G., Staden, R., & Klug, A. (1978) Nature (London) 276, 362-368]. Under low-salt conditions, 20S aggregates are in equilibrium with 4S species and involved in the efficient nucleation of TMV assembly in vitro [Butler, P.J.G. (1984) J. Gen. Virol. 65, 253-279]. We have investigated by sedimentation velocity and near-UV circular dichroism (CD) measurements the structure of 20S aggregates in low salt (I = 0.1 potassium phosphate at pH 7.0 and 20 degrees C) and the aggregates in high salt [0.2 M (NH4)2SO4 in I = 0.1 tris(hydroxymethyl)aminomethane hydrochloride at pH 8.0 and 20 degrees C, close to the conditions under which TMVP crystallizes as disk aggregates]. At high salt, we observe structures (presumably stacks of disks) having s20,w values around 40, 45, and 50 S, but not the 20S species present in low-salt buffers. The near-UV CD spectrum of 20S aggregates has been obtained for the first time, using computer techniques, from the spectra of the 4S-20S equilibrium mixture and the 4S species. This spectrum of 20S aggregates differs dramatically from that of the stacks of disks examined at both high and low salt (into which the stacks can be returned by dialysis), indicating that the difference is not a solvent effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sedimentation equilibrium measurements of the intermediate-size tobacco mosaic virus protein polymers

Short-column sedimentation equilibrium methods have been applied for the first time to tobacco mosaic virus (TMV) protein (0.1 M ionic strength orthophosphate) at pH 6.5 and at pH 7.0 to estimate molecular weights. Previous sedimentation velocity experiments at pH 6.5, 20 degrees C have led to the conclusion that the major boundary with an S0(20),w value of 24.4 +/- 0.1 S consists of a distribution of polymers which are mainly three-turn, 48-51-subunit helical rod aggregates. The directly measured z-average molecular weights together with sedimentation velocity data are entirely consistent with this assignment of a three-turn aggregate. Molecular weights have also been determined under two conditions where a large mass fraction of the protein sediments with an S0(20),w value of 20.3 +/- 0.2 S. At pH 6.5, 6-8 degrees C, the aggregates in this boundary are metastable and correspond to 50-60% of the preparation. At pH 7.0, 20 degrees C at equilibrium, 65-75% of the protein sediments at 20.3 S. The 20.3S boundary is very similar under both conditions and is interpreted as being composed of a distribution of protein aggregates centered about 39 +/- 2 subunits. This result is important in the interpretation of previous kinetic measurements of TMV self-assembly. The current view is that the 34-subunit structure of TMV protein, in the form of a cylindrical disk which is made up of two 17-subunit layers and has been characterized in single-crystal X-ray diffraction studies, plays a central role in the initial binding steps with RNA. The present results are not consistent with the view that there is a significant concentration of the TMV protein disk structure in solution under the usual conditions of TMV self-assembly.     

Disk aggregates of tobacco mosaic virus protein in solution: electron microscopy observations

Previous studies of the coat protein of tobacco mosaic virus (TMVP) have shown that TMVP presumably exists as linear stacks of two-ring cylindrical disks in the 0.7 M ionic strength buffer used for crystallizing the disks for X-ray diffraction studies [Raghavendra, K., Adams, M.L., & Schuster, T.M. (1985) Biochemistry 24, 3298-3304]. The spectroscopic and sedimentation studies of solutions of TMVP under these crystallizing conditions have demonstrated a long-term metastability of these disk aggregates when they are placed in 0.1 M ionic strength buffers, as are used for reconstituting tobacco mosaic virus from TMVP and viral RNA. The present work describes an electron microscopic study of TMVP disk aggregates under the same solution conditions employed in the previous spectroscopic and sedimentation studies. The results show that in the pH 8.0 0.7 M ionic strength crystallization buffer TMVP exists as stacks of disks which range in size from about 6 to 24 layers, corresponding to 3-12 2-layer disk aggregates having 17 subunits per layer. These TMVP aggregates persist in a metastable form in 0.1 M ionic strength virus reconstitution buffer with no apparent changes in structure of the stacked disks. The results are consistent with the conclusions of the solution physical-chemical studies which suggest that the disk structure may not be related to the 20S TMVP aggregate that is the nucleation species in virus     

Studies on the mechanism of assembly of tobacco mosaic virus.

Sedimentation and proton binding studies on the endothermic self-association of tobacco mosaic virus (TMV) protein indicate that the so-called "20S" sedimenting protein is an interaction system involving at least the 34-subunit two-turn yield cylindrical disk aggregate and the 49-subunit three-turn helical rod. The pH dependence of this overall equilibrium suggests that disk formation is proton-linked through the binding of protons to the two-turn helix which is not present as significant concentrations near pH 7. There is a temperature-induced intramolecular conformation change in the protein leading to a difference spectrum which is complete in 5 x 10(-6) s at pH 7 and 20 degrees C and is dominated at 300 nm by tryptophan residues. Kinetics measurements of protein polymerization, from 10(-6) to 10(3) s, reveal three relaxation processes at pH 7.0, 20 degrees C, 0.10 M ionic strength K (H) PO4. The fastest relaxation time is a few milliseconds and represents reactions within the 4S protein distribution. The second fastest relaxation is 50-100 x 10(-3) s and represents elementary polymerization steps involved in the formation of the approximately 20 S protein. Analysis of the slowest relaxation, approximately 5 x 10(4) s, suggests that this very slow formation of approximately 20 S protein may be dominated by some first order process in the overall dissociation of approximately 20S protein. Sedimentation measurements of the rate of TMV reconstitution, under the same conditions, show by direct measurements of 4S and approximately 20S incorporation at various 4S to approximately 20S weight ratios that the relative rate of approximately 20S incorporation decreases almost linearly, from 0 to 50% 4S. There appears to be one or more regions of TMV-RNA, approximately 1-1.5 kilobases long, which incorporates approximately 20S protein exclusively. Solutions of approximately 95-100% approximately 20S protein have been prepared for the first time and used for reconstitution with RNA. Such protein solutions yield full size TMV, but at a slower rate than if 4S protein is added. Thus the elongation reaction in TMV assembly, following nucleation with approximately 20S protein, is not exclusively dependent upon the presence of either 4S or approximately 20S protein aggregates. The initial, maximum, rate of reconstitution increases about threefold when the protein composition is changed from 5% to 30% 4S protein, at constant total protein concentration at pH 7.0, 20 degrees C in 0.10 M ionic strength K (H)PO4. The probable binding frame at the internal assembly nucleation site of TMV-RNA has been determined by measuring the association constants for the binding of various trinucleoside diphosphates to helical TMV protein rods. The -CAG-AAG-AAG-sequence at the nucleation site is capable of providing at least 10-14 kcal/mol of sites of binding free energy for the nucleation event in TMV self-assembly.     

Structure and function of disk aggregates of the coat protein of tobacco mosaic virus

Experiments have been carried out on the coat protein of tobacco mosaic virus (TMVP) to test for the occurrence of the previously postulated RNA-induced direct switching, during in vitro assembly of tobacco mosaic virus (TMV), of the subunit packing from the cylindrical bilayer disk to the virus helical arrangement. No evidence was found for such RNA-induced switching and no evidence for the direct participation of the bilayer disk in either the nucleation or elongation phases of the in vitro virus assembly. Instead, virus assembly proceeds by an initiation step involving the binding of the RNA to the previously characterized two-plus turn helical aggregate that is formed from small oligomers of subunits. However, a bilayer disk, which has been characterized in high ionic strength crystals, has been observed in low ionic strength virus assembly solutions only as a transient species upon depolymerization of dimers of bilayer disks formed in solution at high ionic strength, and not as an equilibrium species of TMVP.     

Self-assembly of tobacco mosaic virus: the role of an intermediate aggregate in generating both specificity and speed

The tobacco mosaic virus (TMV) particle was the first macromolecular structure to be shown to self-assemble in vitro, allowing detailed studies of the mechanism. Nucleation of TMV self-assembly is by the binding of a specific stem-loop of the single-stranded viral RNA into the central hole of a two-ring sub-assembly of the coat protein, known as the 'disk'. Binding of the loop onto its specific binding site, between the two rings of the disk, leads to melting of the stem so more RNA is available to bind. The interaction of the RNA with the protein subunits in the disk cause this to dislocate into a proto-helix, rearranging the protein subunits in such a way that the axial gap between the rings at inner radii closes, entrapping the RNA. Assembly starts at an internal site on TMV RNA, about 1 kb from its 3'-terminus, and the elongation in the two directions is different. Elongation of the nucleated rods towards the 5'-terminus occurs on a 'travelling loop' of the RNA and, predominantly, still uses the disk sub-assembly of protein subunits, consequently incorporating approximately 100 further nucleotides as each disk is added, while elongation towards the 3'-terminus uses smaller protein aggregates and does not show this 'quantized' incorporation.     

Oligonucleotide binding to the coat protein disk of tobacco mosaic virus. Possible steps in the mechanism of assembly

Binding of the oligoribonucleotides AAG, AAGAAG and AAGAAGUUG to the disk aggregate of tobacco mosaic virus coat protein has been studied in solution under conditions favourable for virus assembly. The two longer oligomers bind strongly with Kd around 1 microM, approach complete saturation of binding sites and cause the formation of long, nicked helical rods resembling the virus. It is suggested that the binding of these oligomers, with sequences chosen from the assembly origin of the viral RNA, simulates the tobacco mosaic virus assembly process. No binding could be detected for AAG, indicating that chain length is a crucial determinant in the interaction. The binding of AAGAAG to coat protein crystals is very much weaker than that observed in solution, and the crystals crack at high oligomer concentrations. The corresponding oligodeoxyribonucleotide, d(AAGAAG), shows no binding to the protein in solution; the interaction is extremely specific for RNA.     

Assembly mechanism of tobacco mosaic virus particle from its ribonucleic acid and protein

Summary The reconstitution process of an infectious tobacco mosaic virus particle from its RNA and protein consists of two steps, formation of the initial complex and growth of the helical rod, the former is the rate limiting step. The protein aggregate, having about 20–30 S, is needed for the formation of the initial complex with 5-end of tobacco mosaic virus RNA. The elongation reaction from the initial complex proceeds even under conditions where both the reconstitution reaction and the formation of 20–30 S protein aggregates do not take place. This indicates that the growth of the helical rod proceeds by stepwise additions of protein subunits or 4 S aggregates. A possible model for assembly process of tobacco mosaic virus particle is presented.     

An extended secondary structure model for the TMV assembly origin, and its correlation with protection studies and an assembly defective mutant

Recognition of the unique internal assembly origin on tobacco mosaic virus (TMV) RNA by the disk aggregate of the viral coat protein probably involves an extended region of the RNA (larger than that coated by a single disk) folded into a specific conformation. A secondary structure model is proposed for the RNA preferentially coated by limiting amounts of coat protein disks on the basis of partial nuclease digestion data. Part of this sequence can form three symmetrically spaced hairpins with marginally stable base paired sequences at the tips of the stems. The pattern of progressive protection of the RNA from nuclease attack during assembly suggests that these three hairpins are successively coated by the first three disks to add. The spacing of these hairpins is identical to that of three hairpins in the pseudo assembly origin (part of the coat protein gene homologous to the assembly origin). In Ni 2519, a TMV mutant whose assembly is defective at high temperature because it can no longer discriminate between the true and pseudo assembly origins, a point mutation has occurred near the tip of the third metastably base paired stem of the true assembly origin which would disrupt its structure and alter one copy of a repeated heptanucleotide. This suggests an important role for the ordered and cooperative recognition of successive loops in determining the specificity of assembly.     

Mechanism of self-assembly of tobacco mosaic virus protein. I. Nucleation-controlled kinetics of polymerization.

The polymerization of tobacco mosaic virus protein has been found to proceed through metastable states under conditions where initially one of the two polymerization-linked protons is bound. These metastable polymers have been characterized and are found to be helical rods, which resemble the structure of equilibrium helical rods that form when both polymerization-linked protons are bound. At pH 6.5 and 20 °C the true equilibrium distribution of these helical rods has been shown to consist of sedimenting species that are much smaller, 24 to 34 S, than described previously, 100 to 200 S. The larger, non-equilibrium rods are produced by an overshoot in polymerization that results from the slow formation of 20 S nuclei followed by a very rapid elongation reaction. Generally, this sequence of rate processes is sensitive to the rate at which a reaction is initiated. In the present case it is the rate of heating or the rate of change of the pH that determines the reaction path and therefore the rate of attainment of equilibrium. In addition to the formation of metastable helical rods during polymerization overshoot, metastable 20 S aggregates can form when either equilibrium or non-equilibrium helical rods are depolymerized by cooling to 5 to 7 °C at pH 6.5. These 20 S aggregates are presumably two-turn disks or helices and can serve as nuclei for helical rod formation in subsequent polymerization reactions. Both helical rod and 20 S metastability are extremely sensitive to pH but, under carefully controlled conditions, the metastability is quite reproducible and reproducible nucleation-controlled polymerization kinetics can be observed even when polymerization-depolymerization cycling is carried out between branches of a hysteresis loop. Temperature- or pH-induced polymerization of tobacco mosaic virus protein can be made to proceed by the slow formation of 20 S, two-turn helix, nuclei followed by the rapid addition of one or more species comprising the 4 S protein. These results confirm a previously proposed kinetic mechanism for the non-equilibrium polymerization reaction (Scheele &; Schuster, 1974).     

Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein

Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris

The hypersensitivity resistance response directed by the N' gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild-type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild-type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4 degrees C. Viral reconstitution kinetics at 20 degrees C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus.     

Studies on the assembly of a spherical plant virus. I. States of aggregation of the isolated protein

The aggregates formed at equilibrium by purified protein from cowpea chlorotic mottle virus have been characterized on the basis of their sedimentation behaviour and appearance in the electron microscope. Between pH 3.5 and 7.5, at ionic strengths greater than 0.2, most of the protein is found in aggregates sedimenting at either 3 S or 50 S. The 50 S aggregate is identified as the reassembled capsid of cowpea ehlorotic mottle virus. Decreasing the ionic strength favours the formation of multi-shelled particles. Below pH 5.5 single- and multishelled particles predominate, while above this pH most of the protein sediments at 3 S.Varying the temperature from 5 °C to 20 °C has little effect on the equilibrium proportions of aggregates although some real differences can be detected. Ionic strength is not as important a variable as pH in determining which protein forms are present (but increasing ionic strength does result in a steady decrease in the proportion of protein in the multi-layer aggregates). The dependence of the equilibrium upon protein concentration shows that capsid formation is a quasi-crystallization: beyond a certain total protein concentration the concentration of 3 S aggregate remains at this “critical” concentration and all further protein goes into 50 S capsid. In addition to shells and variations upon shells, tubes and hexagonal nets of protein subunits have occasionally been seen with the electron microscope.     

Temperature dependence of the structure of aggregates of tobacco mosaic virus protein at pH 7.2. Static synchrotron small-angle X-ray scattering

The small-angle X-ray scattering (SAXS) method using a synchrotron radiation source was applied to the study of the self-aggregation process of tobacco mosaic virus protein (TMVP) at a concentration of 5.0 or 12.0 mg ml-1 in 50 mM or 100 mM-phosphate buffer (ionic strengths approx. 0.1 and 0.2, respectively) at pH 7.2 in the temperature region of 4.8 to 25.0 degrees C. This paper presents the results of static measurements of SAXS. Sedimentation velocity experiments were performed simultaneously under the same conditions. These results are qualitatively parallel to those of the SAXS measurements, although the size of stacked disks derived from the SAXS measurements is larger than that derived from the sedimentation experiments, suggesting a change in the equilibrium conditions in the centrifugal field. Qualitative analysis of the SAXS data with model simulation calculations implies that the aggregation of TMVP consists of two steps: (1) the aggregation of A-protein comprising a few subunits to form double-layered disks; and (2) the random polymerization of double-layered disks by disk-stacking. Increase in temperature, ionic strength or protein concentration induced TMVP to polymerize to form a double-layered disk or a quadruple-layered short rod with consumption of A-proteins, accompanied by a small number of multi-layered short rods. The SAXS results indicate that the A-protein and the multilayered short rods are polydisperse with respect to size and shape, i.e. the mixture of A-protein, double-layered disks and multi-layered short rods coexists in the equilibrium state without pressure-induced partial dissociation of TMPV as observed during normal ultracentrifugation, and even under solution conditions in which the formation of double-layered disks or higher-order aggregates is favored.     

Structures and roles of the polymorphic forms of tobacco mosaic virus protein: VI. Assembly of the nucleoprotein rods of tobacco mosaic virus from the protein disks and RNA

The assembly of tobacco mosaic virus involves a preformed protein aggregate, the disk, which consists of two rings each of 17 protein subunits, as the sole protein source. The kinetics of this assembly have been studied, using both tobacco mosaic virus RNA, which causes a rapid initiation and so enables growth to be studied, and also polyadenylic acid, with which initiation is slowed down and thus can be partially resolved from growth. Two disks interact with a special nucleotide sequence at the 5′-hydroxyl end of a single tobacco mosaic virus RNA molecule to initiate the formation of the viral nucleoprotein helix, which then grows by the addition of further disks. All of the subunits from these further disks are incorporated into the helix, so that growth proceeds by the co-operative addition of 34 subunits at a time. Under the conditions used, rearrangement of each disk takes about six seconds, giving a total time for the growth of a complete virus particle of just over six minutes.     

The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis

The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.     

States of aggregation of the dahlemense strain of tobacco mosaic virus protein and their relation to crystal formation.

The aggregation of the protein of the dahlemense strain of tobacco mosaic virus has been studied by electron microscopy and ultracentrifugation. The aggregates formed are similar to those formed by the vulgare strain, although the particular conditions for their formation are often rather different. Helix formation by dialysis of A protein at pH 8 to acid pH is much more efficient if an intermediate step at pH 7 is introduced. The 20 S particle or two-layer disk is stable over a wide range of pH and ionic strength values. There is no tendency to form short stacks of disks at high ionic strength; instead, 30 S particles are formed that correspond to a pair of interlocked disks giving a “figure-of-eight” appearance in electron micrographs. These particles appear to be the “building blocks” of the protein crystal.     

Circular dichroism and sedimentation studies on the reconstitution of tobacco mosaic virus

Reconstitution of tobacco mosaic virus from its constituents, the coat protein and RNA, was investigated by means of ultracentrifugation and circular dichroism measurement. Tobacco mosaic virus protein forms a 20S double-layer disc under conditions favorable for tobacco mosaic virus reconstitution. Dibromination of the tyrosine 139 residue of tobacco mosaic virus protein prevents formation of the 20S disc.Acidification of the tobacco mosaic virus protein solution causes 20S discs to polymerize into long helical rods. Changes in the CD spectra of tobacco mosaic virus protein in the near-ultraviolet region suggest that stacking of the aromatic sidechains of amino acid residues stabilizes the helical rod. The dibrominated tobacco mosaic virus protein also has the ability of rod elongation under acidic condition. CD studies reveal that assembly of tobacco mosaic virus particles from its constituents is stabilized by the stacking effect between the base residues of RNA and the aromatic residues of tobacco mosaic virus protein.Cucumber green mottle mosaic virus protein, which acts as a substituent for tobacco mosaic virus protein in tobacco mosaic virus reconstitution, was also investigated.     

Refined atomic model of the four-layer aggregate of the tobacco mosaic virus coat protein at 2.4-A resolution.

Previous x-ray studies (2.8-A resolution) on crystals of tobacco mosaic virus coat protein grown from solutions containing high salt have characterized the structure of the protein aggregate as a dimer of a bilayered cylindrical disk formed by 34 chemically identical subunits. We have determined the crystal structure of the disk aggregate at 2.4-A resolution using x-ray diffraction from crystals maintained at cryogenic temperatures. Two regions of interest have been extensively refined. First, residues of the low-radius loop region, which were not modeled previously, have been traced completely in our electron density maps. Similar to the structure observed in the virus, the right radial helix in each protomer ends around residue 87, after which the protein chain forms an extended chain that extends to the left radial helix. The left radial helix appears as a long alpha-helix with high temperature factors for the main-chain atoms in the inner portion. The side-chain atoms in this region (residues 90-110) are not visible in the electron density maps and are assumed to be disordered. Second, interactions between subunits in the symmetry-related central A pair have been determined. No direct protein-protein interactions are observed in the major overlap region between these subunits; all interactions are mediated by two layers of ordered solvent molecules. The current structure emphasizes the importance of water in biological macromolecular assemblies.     

Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH.

The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.