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1.
Wikoff WR Conway JF Tang J Lee KK Gan L Cheng N Duda RL Hendrix RW Steven AC Johnson JE 《Journal of structural biology》2006,153(3):300-306
The bacteriophage HK97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. Immature empty capsids, termed Prohead II, assemble in vivo in an Escherichia coli expression system. Maturation of these particles may be induced in vitro, converting them into Head II capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. One method of in vitro maturation requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a beta-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained. 相似文献
2.
The structure of the capsid of bacteriophage HK97 has been solved at various stages of maturity by crystallography and cryo-electron microscopy, and has been reported previously in the literature. Typically the capsid assembles through polymerization and maturation processes. Maturation is composed of proteolytic cleavages to the precursor capsid (called Prohead II), expansion triggered by DNA packaging (in which the largest conformational changes of the capsid appear), and covalent cross-links of neighboring subunits to create the mature capsid called Head II. We apply a coarse-grained elastic network interpolation (ENI) to generate a feasible pathway for conformational change from Prohead II to Head II. The icosahedral symmetry of the capsid structure offers a significant computational advantage because it is not necessary to consider the whole capsid structure but only an asymmetric unit consisting of one hexamer plus an additional subunit from an adjacent pentamer. We also analyze normal modes of the capsid structure using an elastic network model which is also subject to symmetry constraints. Using our model, we can visualize the smooth evolution of capsid expansion and revisit in more detail several interesting geometric changes recognized in early experimental works such as rigid body motion of two compact domains (A and P) with two refolding extensions (N-arm and E-loop) and track the approach of the two particular residues associated with isopeptide bonds that make hexagonal cross-links in Head II. The feasibility of the predicted pathway is also supported by the results of our normal mode analysis. 相似文献
3.
Gan L Conway JF Firek BA Cheng N Hendrix RW Steven AC Johnson JE Duda RL 《Molecular cell》2004,14(5):559-569
Radical structural changes drive the maturation of the capsid of HK97, a lambda-like, dsDNA bacteriophage of Escherichia coli. These include expansion from approximately 560 to approximately 660 A in diameter, metamorphosis from a round to an angular shape, and formation of covalent crosslinks between adjacent capsomers. Analogous transformations also occur in unrelated viruses and protein complexes. We find that expansion and crosslinking happen concurrently during maturation at low pH. Expansion causes residues on three different subunits to move up to 35 A to form 420 active sites that each catalyze the formation of a lysine-asparagine crosslink between adjacent subunits, making crosslink formation an indirect reporter of structural change. Intermediate crosslinking patterns support a previously proposed model of expansion, while hydrophobic properties aid in distinguishing discrete intermediates. A structure derived from cryo-EM images reveals the free intermediate conformation of penton arms, supporting our model for coordinated movement of hexons and pentons on the capsid lattice. 相似文献
4.
Head assembly in the double-stranded DNA coliphage HK97 involves initially the formation of the precursor shell Prohead I from approximately 420 copies of a 384-residue subunit. This is followed by proteolytic removal of residues 2-103 to create Prohead II, and then reorganization and expansion of the shell lattice and covalent cross-linking of subunits make Head II. Here, we report and structurally interpret solution Raman spectra of Prohead I, Prohead II, and Head II particles. The Raman signatures of Prohead I and Prohead II indicate a common alpha/beta fold for residues 104-385, and a strongly conserved tertiary structure. The Raman difference spectrum between Prohead I and Prohead II demonstrates that the N-terminal residues 2-103 (Delta-domain) form a predominantly alpha-helical fold devoid of beta-strand. The conformation of the Delta-domain in Prohead I thus resembles that of the previously characterized scaffolding proteins of Salmonellaphage P22 and Bacillus phage phi29 and suggests an analogous architectural role in mediating the assembly of a properly dimensioned precursor shell. The Prohead II --> Head II transition is accompanied by significant reordering of both the secondary and tertiary structures of 104-385, wherein a large increase occurs in the percentage of beta-strand (from 38 to 45%), and a marginal increase is observed in the percentage of alpha-helix (from 27 to 31%). Both are at the expense of unordered chain segments. Residue environments affected by HK97 shell maturation include the unique cysteine (Cys 362) and numerous tyrosines and tryptophans. The tertiary structural reorganization is reminiscent of that observed for the procapsid --> capsid transformation of P22. The Raman signatures of aqueous and crystalline Head II reveal no significant differences between the crystal and solution structures. 相似文献
5.
Critical Salt Bridges Guide Capsid Assembly,Stability, and Maturation Behavior in Bacteriophage HK97
Ilya Gertsman Chi-Yu Fu Rick Huang Elizabeth A. Komives John E. Johnson 《Molecular & cellular proteomics : MCP》2010,9(8):1752-1763
HK97 is a double-stranded DNA bacteriophage that undergoes dramatic conformational changes during viral capsid maturation and for which x-ray structures, at near atomic resolution, of multiple intermediate and mature capsid states are available. Both amide H/2H exchange and crystallographic comparisons between the pre-expanded Prohead II particles and the expanded Head II of bacteriophage HK97 revealed quaternary interactions that remain fixed throughout maturation and appear to maintain intercapsomer integrity at all quasi- and icosahedral 3-fold axes. These 3-fold staples are formed from Arg and Glu residues and a metal binding site. Mutations of either Arg-347 or Arg-194 or a double mutation of E344Q and E363A resulted in purification of the phage in capsomer form (hexamers and pentamers). Mutants that did assemble had both decreased thermal stability and decreased in vitro expansion rates. Amide H/2H exchange mass spectrometry showed that in the wild type capsid some subunits had a bent “spine” helix (highly exchanging), whereas others were straight (less exchanging). Similar analysis of the never assembled mutant capsomers showed uniform amide exchange in all of these that was higher than that of the straight spine helices (characterized in more mature intermediates), suggesting that the spine helix is somewhat bent prior to capsid assembly. The result further supports a previously proposed mechanism for capsid expansion in which the delta domains of each subunit induce a high energy intermediate conformation, which now appears to include a bent helix during capsomer assembly.The viral capsid, particularly in double-stranded DNA bacteriophage, requires a highly stable macromolecular structure capable of encapsulating genome at near liquid crystalline density. Viral capsids are composed of hundreds to thousands of individual subunits that efficiently assemble into a closed capsid form often of a highly symmetrized icosahedral geometry, avoiding kinetic traps that would result in increased off-pathway assemblies. Recent studies have proposed that capsid assembly is mediated by weak intersubunit interactions that nucleate larger assembly intermediates, resulting in a considerably more stable capsid form due to a favorable geometry with a more constrained network of interactions. Measurements in systems such as cowpea chlorotic mottle virus, hepatitis B virus, and the bacteriophages P22 and HK97 have estimated the association energy of the initial assembly interaction between two subunits at 2–5 kcal/mol, which is seemingly low for a robust assembly product (1–5). An entropically driven process based on burial of hydrophobic surfaces was considered the driving force for the initial weak interactions with subsequent nucleation and elongation reactions leading to assembly of the full capsid (2, 6). Most complex viruses undergo a staged assembly process involving conformational transitions that occur after the initial assembly of a procapsid (7). The process is known as virion maturation. The interplay between interactions necessary for the initial assembly of capsomers into the procapsid and those that facilitate capsid maturation have been poorly understood, but recent crystal structures of procapsid and mature capsid states of HK97 allowed us to evaluate the structural properties that may facilitate maturation.HK97 is an amenable system for the study of capsid assembly and maturation. Symmetric procapsid particles can be assembled in Escherichia coli with the expression of just two gene products, gp4 (protease) and gp5 (capsid subunit). Maturation can then be followed in vitro by lowering the pH or chemically perturbing the procapsids. Unlike bacteriophages such as P22 that assemble their capsids directly from individual monomeric subunits, HK97 subunits initially assemble into capsomers composed of six-subunit (hexamers) or five-subunit (pentamers) oligomers. Twelve pentamers and 60 hexamers then assemble to form an icosahedral capsid with a triangulation number of 7 laevo, although a portal complex substitutes one of the pentamers during in vivo assembly. Residues 2–103 at the N terminus of the subunit, referred to as the delta domain, is thought to serve the same role as the scaffolding proteins identified for other phage in the assembly process (8). Capsomers then assemble, packaging the protease (gp4), to form the initial procapsid, Prohead I (P-I).1 If the expression is done without the protease or with an inactive (by mutation) protease, this step is reversible (Fig. 1). The equilibrium of this assembly can be controlled in vitro with specific buffers and concentrations that favor either the capsomer or the capsid form (9). Expression with an active protease leads to proteolysis of the delta domains in the assembled P-I state followed by autodigestion of the protease and diffusion of the fragments from the particle. P-I then undergoes subtle structural adjustments, resulting in the Prohead II state composed entirely of the cleaved gp5* subunits (10, 11). At this stage of assembly in vivo, concatameric double-stranded DNA is packaged through a portal complex (composed of gp3 subunits) that fits into a single 5-fold vertex of the capsid. We used an HK97 construct that lacks gp3, so the purified Prohead II capsid is icosahedrally symmetric and cannot package DNA. Purified P-II can be matured in vitro using low pH and other chemical perturbation methods. During maturation, conformational changes in the subunits and their interactions result in large scale expansion and morphological changes in the capsid. The diameter of the capsid shell increases from 540 Å in P-II to 660 Å in Head II (H-II), the fully expanded particle form (12, 13). Intermediate particle forms can be trapped during the expansion and were previously characterized with a variety of biophysical techniques including cryo-EM microscopy (14, 15), x-ray crystallography (12, 13, 16), and small angle x-ray scattering (16–18). During the expansion process, self-catalyzed covalent cross-links are formed through isopeptide bond formation between Lys-169 and Asn-356 of different subunits situated on adjacent capsomers (19). The reaction is promoted by Glu-363, which is adjacent to the bonding residues and functions as a proton acceptor. Cross-linking during maturation was previously shown by differential scanning calorimetry (DSC) to greatly enhance the thermal stability of HK97 (5). In addition to covalent bonding, the H-II has significantly more buried surface area than P-II as seen in the highly intercalated intersubunit interactions depicted in the previous 3.44-Å structure of Head II (13, 20). A cross-link-defective mutant, K169Y, stills undergoes particle expansion, reaching the penultimate particle form, termed Head I (H-I), which has nearly identical conformations of hexamer capsomers but less extruded pentamers than H-II (16). H-I was used for all H/2H exchange studies instead of H-II because the cross-links in H-II dramatically affect the efficiency of proteolysis required for the mass spectrometry-based experiment (12, 20).Open in a separate windowFig. 1.HK97 assembly and expansion pathway. The schematic diagram depicts the assembly and expansion of HK97 in an E. coli expression system lacking the portal protein and other machinery required for genome packaging. 42-kDa subunits assemble into hexamer and pentamer capsomers, which then assemble into an initial icosahedral procapsid shell, P-I. Proteolytic cleavage of the delta domain of each subunit results in the formation of the metastable intermediate form P-II, which is able to undergo in vitro maturation when perturbed by various chemical agents. WT expansion proceeds through EI, balloon, and ultimately H-II forms, an expansion process that involves covalent cross-linking. K169Y mutant P-II proceeds through EI to the H-I form without any cross-linking occurring. Other than the lack of cross-links, H-I is identical to balloon.It was hypothesized that for highly intercalated mature capsid forms such as that seen in bacteriophage HK97 early procapsid intermediates are necessary for initial positioning of subunits before conformational changes can facilitate a protein architecture with increased stability. We recently showed with amide H/2H exchange and crystallographic comparisons between the pre-expanded P-II particles and the mature H-II that maturation is probably guided by tertiary structure twisting and secondary structure changes around a fixed set of intercapsomer interactions that surround all quasi- and icosahedral 3-fold axes in the capsid shell (12). The major interactions that appear to facilitate these “3-fold staples” include two sets of salt bridges and a putative metal binding site (Fig. 2). The salt bridge interactions are between residues Glu-344 and Arg-194 and between residues Glu-363 and Arg-347. Glutamate 363 serves dual roles as it is involved in both a salt bridge with Arg-347 and serves as a proton acceptor that catalyzes the isopeptide bond formation (21). The metal binding site is formed by 3-fold related glutamates at position 348 interacting with a sphere of electron density at high σ level in the P-II crystal structure (12). Although comparable density for metal ions is not present at the equivalent position in crystal structures of the late intermediates, the positions of the glutamates are nearly identical, indicating a stable interaction with some mechanism for neutralizing the negative charge repulsion. In contrast to the near identical conformations of the residues at the 3-fold interface, the rest of the subunit was shown to undergo a large scale twisting motion, causing hinging in all three P-domain β-strands (see Fig. 8A for domain nomenclature). These data imply that interactions at the 3-fold interface may be crucial in assembling the capsid from individual capsomers as well as providing a fixed point from which subunits bend while maintaining intercapsomer contacts.Open in a separate windowFig. 2.Importance of 3-fold intercapsomer contacts. A, P-II capsid from previously solved 3.65-Å crystal structure rendered in low resolution in chimera. Two hexon subunits (subunits a and f, yellow and green, respectively) and one penton subunit (orange) that form a quasi-3-fold interaction are shown as ribbons. B, zoomed in view of quasi-3-fold interaction between the two hexamer subunits and one pentamer subunit as highlighted in A. The view is from inside the capsid, 180° rotated from the view shown in A. Residues involved in salt bridges as well as a putative metal binding site (Glu-348) are labeled accordingly. C, table identifying various mutations made to perturb 3-fold contacts. The phenotypes following protein expression are identified. Mutants are distinguished as to whether they were purified as capsids or capsomers (hexamers and pentamers) following protein expression. Data for the Glu-363 mutants are from Dierkes et al. (21).Open in a separate windowFig. 8.Solvent accessibility of R347N capsomer spine helix. A, subunit C of Prohead II is shown with the major domains labeled. Residues 206–216 of the spine helix are colored orange. B, mass envelopes for P-II and H-I particle forms as well as the R347N capsomers following 5 min of exchange. The top spectrum is non-deuterated P-II. C, H/2H exchange results of the residues colored orange are plotted for the R347N capsomers (orange curve) and compared with the solvent accessibility curves for the same fragment in the P-II capsid state, EI, and the nearly mature H-I capsid form. D, the solvent accessibility of the same spine helix fragment is shown for both the R347N capsomers and WT capsomers that were disassembled from the P-I state.Here we confirmed this role for the 3-fold interactions by mutagenesis of relevant residues and characterized the resulting assembly products, thermal stabilities, and maturation kinetics. Some of the mutants did not assemble into particles following the formation of capsomers (e.g. R347N). Capsomers were then purified, and the amide exchange of the spine helices was analyzed with H/2H exchange coupled to mass spectrometry (22–24). Previous data illustrated a direct correlation between increased H/2H exchange and an increased bend in the helix conformation (12, 20). Amide exchange of the spine helix in the mutant capsomers was compared with previously characterized particle forms as well as P-I and WT capsomers disassembled from P-I. 相似文献
6.
Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization
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Ross PD Cheng N Conway JF Firek BA Hendrix RW Duda RL Steven AC 《The EMBO journal》2005,24(7):1352-1363
In HK97 capsid maturation, structural change ('expansion') is accompanied by formation of covalent crosslinks, connecting residue K169 in the 'E-loop' of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead-II passes through three expansion intermediate (EI) states en route to the end state, Head-II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild-type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (deltaTp, +11 degrees C). EI-II is the earliest capsid to form crosslinks. Cryo-electron microscopy shows that for both wild-type and K169Y EI-II, most E-loops are in the 'up' position, 30 A from the nearest N356: thus, crosslinking in EI-II represents capture of mobile E-loops in 'down' positions. At pH 4, most K169Y capsids remain as EI-II, whereas wild-type capsids proceed to EI-III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism. 相似文献
7.
The G-loop is a 10-residue glycine-rich loop that protrudes from the surface of the mature bacteriophage HK97 capsid at the C-terminal end of the long backbone helix of major capsid protein subunits. The G-loop is essential for assembly, is conserved in related capsid and encapsulin proteins, and plays its role during HK97 capsid assembly by making crucial contacts between the hill-like hexamers and pentamers in precursor proheads. These contacts are not preserved in the flattened capsomers of the mature capsid. Aspartate 231 in each of the ~ 400 G-loops interacts with lysine 178 of the E-loop (extended loop) of a subunit on an adjacent capsomer. Mutations disrupting this interaction prevented correct assembly and, in some cases, induced abnormal assembly into tubes, or small, incomplete capsids. Assembly remained defective when D231 and K178 were replaced with larger charged residues or when their positions were exchanged. Second-site suppressors of lethal mutants containing substitution D231L replaced the ionic interaction with new interactions between neutral and hydrophobic residues of about the same size: D231L/K178V, D231L/K178I, and D231L/K178N. We conclude that it is not the charge but the size and shape of the side chains of residues 178 and 231 that are important. These two residues control the geometry of contacts between the E-loop and the G-loop, which apparently must be precisely spaced and oriented for correct assembly to occur. We present a model for how the G-loop could control HK97 assembly and identify G-loop-like protrusions in other capsid proteins that may play analogous roles. 相似文献
8.
Lee KK Gan L Tsuruta H Hendrix RW Duda RL Johnson JE 《Journal of molecular biology》2004,340(3):419-433
Bacteriophage capsids are a striking example of a robust yet dynamic genome delivery vehicle. Like most phages, HK97 undergoes a conformational maturation that converts a metastable Prohead into the mature Head state. In the case of HK97, maturation involves a significant expansion of the capsid and concomitant cross-linking of capsid subunits. The final state, termed Head-II, is a 600 angstroms diameter icosahedral structure with catenated subunit rings. Cryo-EM, small angle X-ray scattering (SAXS), and biochemical assays were used previously to characterize the initial (Prohead-II) and final states (Head-II) as well as four maturation intermediates. Here we extend the characterization of the acid-induced expansion of HK97 in vitro by monitoring changes in intrinsic fluorescence, circular dichroism (CD), and SAXS. We find that the greatest changes in all observables occur at an early stage of maturation. Upon acidification, fluorescence emissions from HK97 exhibit a blueshift and decrease in intensity. These spectral changes reveal two kinetic phases of the expansion reaction. The early phase exhibits sensitivity to pH, increasing in rate nearly 200-fold when acidification pH is lowered from 4.5 to 3.9. The second, slower phase reported by fluorescence is relatively insensitive to pH. Time-resolved SAXS experiments report an increase in overall particle dimension that parallels the fluorescence changes for the early phase. Native agarose gel assays corroborated this finding. By contrast, probes of CD at far-UV indicate that secondary structural changes precede the early expansion phase reported by SAXS and fluorescence. Based on the crystallographic structure of Head-II and the pseudo-atomic model of Prohead-II, we interpret these changes as reflecting the conversion of subunit N-terminal arms (N-arm) from unstructured polypeptide to the mixture of beta-strand and beta-turn observed in the Head-II crystal structure. Refolding of the N-arm may thus represent the conformational trigger that initiates the irreversible expansion of the phage capsid. 相似文献
9.
Xiao B Shi G Gao J Blaszczyk J Liu Q Ji X Yan H 《The Journal of biological chemistry》2001,276(43):40274-40281
The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-A resolution with a crystallographic R factor of 0.191. The solution structure of HPPK in complex with Mg(2+) and beta,gamma-methyleneadenosine 5'-triphosphate (MgAMPPCP) has been determined using a simulated annealing protocol with 3,523 experimental NMR restraints. The root mean square deviation of the ensemble of 20 refined conformers that represent the solution structure from the mean coordinate set derived from them is 0.74 +/- 0.26 A for all backbone atoms and 0.49 +/- 0.22 A when residues Pro(14), Pro(44)-Gln(50), and Arg(84)-Pro(91) are excluded. Binding of MgADP causes significant changes in the conformation and dynamical property of three loops of HPPK that are involved in catalysis. A dramatic, unusual conformational change is that loop 3 moves away from the active center significantly with some residues moving by >17 A. The binding of MgADP also stabilizes loop 1 and loop 3 but makes loop 2 more mobile. Very similar conformational and dynamical changes are observed in the NMR solution structure of HPPK.MgAMPPCP. The conformational and dynamical changes may play important roles in both substrate binding and product release in the catalytic cycle. 相似文献
10.
Oling F Santos JS Govorukhina N Mazères-Dubut C Bergsma-Schutter W Oostergetel G Keegstra W Lambert O Lewit-Bentley A Brisson A 《Journal of molecular biology》2000,304(4):561-573
Annexins constitute a family of phospholipid- and Ca(2+)-binding proteins involved in a variety of membrane-related processes. The property of several annexins, including annexin A5, to self-organize at the surface of lipid membranes into 2D ordered arrays has been proposed to be functionally relevant in cellular contexts. To further address this question, we investigated the high-resolution structure of annexin A5 trimers in membrane-bound 2D crystals by cryo-electron microscopy (Cryo-EM). A new 2D crystal form was discovered, with p32(1) symmetry, which is significantly better ordered than the 2D crystals reported before. A 2D projection map was obtained at 6.5 A resolution, revealing protein densities within each of the four domains characteristic of annexins. A quantitative comparison was performed between this structure and models generated from the structure of the soluble form of annexin A5 in pseudo-R3 3D crystals. This analysis indicated that both structures are essentially identical, except for small local changes attributed to membrane binding. As a consequence, and contrary to the common view, annexin A5 molecules maintain their bent shape and do not flatten upon membrane binding, which implies either that the four putative Ca(2+) and membrane-binding loops present different types of interaction with the membrane surface, or that the membrane surface is locally perturbed. We propose that the trimerization of annexin A5 molecules is the relevant structural change occurring upon membrane binding. The evidence that 2D arrays of annexin A5 trimers are responsible for its in vitro property of blood coagulation inhibition supports this conclusion. 相似文献
11.
Ross PD Conway JF Cheng N Dierkes L Firek BA Hendrix RW Steven AC Duda RL 《Journal of molecular biology》2006,364(3):512-525
We investigated the thermodynamic basis of HK97 assembly by scanning calorimetry and cryo-electron microscopy. This pathway involves self-assembly of hexamers and pentamers of the precursor capsid protein gp5 into procapsids; proteolysis of their N-terminal Delta-domains; expansion, a major conformational change; and covalent crosslinking. The thermal denaturation parameters convey the changes in stability at successive steps in assembly, and afford estimates of the corresponding changes in free energy. The procapsid represents a kinetically accessible local minimum of free energy. In maturation, it progresses to lower minima in a cascade punctuated by irreversible processes ("locks"), i.e. proteolysis and crosslinking, that lower kinetic barriers and prevent regression. We infer that Delta-domains not only guide assembly but also restrain the procapsid from premature expansion; their removal by proteolysis is conducive to initiating expansion and to its proceeding to completion. We also analyzed the mutant E219K, whose capsomers reassemble in vitro into procapsids with vacant vertices called "whiffleballs". E219K assemblies all have markedly reduced stability compared to wild-type gp5 (DeltaT(p) approximately -7 degrees C to -10 degrees C; where T(p) is the denaturation temperature). As the mutated residue is buried in the core of gp5, we attribute the observed reduction in stability to steric and electrostatic perturbations of the packing of side-chains in the subunit interior. To explain the whiffleball phenotype, we suggest that these effects propagate to the capsomer periphery in such a way as to differentially affect the stability or solubility of dissociated pentamers, leaving only hexamers to reassemble. 相似文献
12.
13.
Huang RK Khayat R Lee KK Gertsman I Duda RL Hendrix RW Johnson JE 《Journal of molecular biology》2011,408(3):541-554
Virus capsid assembly requires recruiting and organizing multiple copies of protein subunits to form a closed shell for genome packaging that leads to infectivity. Many viruses encode scaffolding proteins to shift the equilibrium toward particle formation by promoting intersubunit interactions and stabilizing assembly intermediates. Bacteriophage HK97 lacks an explicit scaffolding protein, but the capsid protein (gp5) contains a scaffold-like N-terminal segment termed the delta domain. When gp5 is expressed in Escherichia coli, the delta domain guides 420 copies of the subunit into a procapsid with T = 7 laevo icosahedral symmetry named Prohead-I. Prohead-I can be disassembled and reassembled under mild conditions and it cannot mature further. When the virally encoded protease (gp4) is coexpressed with gp5, it is incorporated into the capsid and digests the delta domain followed by autoproteolysis to produce the metastable Prohead-II. Prohead-I+P was isolated by coexpressing gp5 and an inactive mutant of gp4. Prohead-I and Prohead-I+P were compared by biochemical methods, revealing that the inactive protease stabilized the capsid against disassembly by chemical or physical stress. The crystal structure of Prohead-I+P was determined at 5.2 Å resolution, and distortions were observed in the subunit tertiary structures similar to those observed previously in Prohead-II. Prohead-I+P differed from Prohead-II due to the presence of the delta domain and the resulting repositioning of the N-arms, explaining why Prohead-I can be reversibly dissociated and cannot mature. Low-resolution X-ray data enhanced the density of the relatively dynamic delta domains, revealing their quaternary arrangement and suggesting how they drive proper assembly. 相似文献
14.
Calcium binding sites in tomato bushy stunt virus visualized by Laue crystallography 总被引:1,自引:0,他引:1
J W Campbell I J Clifton T J Greenhough J Hajdu S C Harrison R C Liddington A K Shrive 《Journal of molecular biology》1990,214(3):627-632
We have collected Laue diffraction data from crystals of tomato bushy stunt virus using the full white X-ray spectrum from the wiggler magnet of the Synchrotron Radiation Source at Daresbury, U.K. A single 24 second exposure of a crystal soaked in EDTA yielded a data set that was 90% complete between 6 and 3.5 A resolution. A large proportion of the data could be measured using an overlap deconvolution routine to separate spatially overlapping reflections in the dense Laue photograph. Reflections with I greater than 2 sigma I (40% of the data set) were subjected to wavelength normalization. A difference Fourier map between these reflections and a monochromatic native set showed, after icosahedral averaging, the three pairs of Ca2+ binding sites related by quasi-symmetry and the movement of a liganding loop in the protein at the A/C subunit interface. The extent and quality of the data obtained from a single Laue photograph of this virus were thus sufficient to detect clearly such small structural alterations. In a second experiment, a Laue photograph was taken from a crystal that was soaked first in EDTA and then in GdCl3. A difference Fourier map between this Laue data set and the Laue data set from the EDTA-soaked crystal showed clearly the Gd3+ sites in the capsid, demonstrating that the Laue technique is a reliable and efficient means for data collection with virus crystals. 相似文献
15.
16.
We present the codimensional principal component analysis (PCA), a novel and straightforward method for resolving sample heterogeneity within a set of cryo-EM 2D projection images of macromolecular assemblies. The method employs PCA of resampled 3D structures computed using subsets of 2D data obtained with a novel hypergeometric sampling scheme. PCA provides us with a small subset of dominating "eigenvolumes" of the system, whose reprojections are compared with experimental projection data to yield their factorial coordinates constructed in a common framework of the 3D space of the macromolecule. Codimensional PCA is unique in the dramatic reduction of dimensionality of the problem, which facilitates rapid determination of both the plausible number of conformers in the sample and their 3D structures. We applied the codimensional PCA to a complex data set of Thermus thermophilus 70S ribosome, and we identified four major conformational states and visualized high mobility of the stalk base region. 相似文献
17.
Conformational changes are thought to play a key role in the function of active protein kinases, although little is known about how these changes relate to the mechanism of phosphorylation. Here we present four high-resolution structures of a single crystal form of Sky1p, a constitutively active serine kinase implicated in yeast RNA processing, each in a different state of nucleotide binding. By comparing the apoenzyme structure to the ADP- and ATP-bound Sky1p structures, we have revealed conformational changes caused by ATP binding or conversion from nucleotide reactant to product. Rotation of the small lobe of the kinase closes the cleft upon binding, allowing the nucleotide to interact with residues from both lobes of the kinase, although some interactions thought to be important for phosphotransfer are missing in the ATP-containing structure. In the apoenzyme, a kinase-conserved phosphate-anchoring loop is in a twisted conformation that is incompatible with ADP and ATP binding, providing a potential mechanism for facilitating ADP release in Sky1p. The nonhydrolyzable ATP analogue AMP-PNP binds in a unique mode that fails to induce lobe closure. This observation, along with comparisons between the two independent molecules in the asymmetric unit of each structure, has provided new molecular details about how the nucleotide binds and induces closure. Finally, we have used mutational analysis to establish the importance of a glycine within the linker that connects the two lobes of Sky1p. 相似文献
18.
Proteins are dynamic molecules, exhibiting structural heterogeneity in the form of anisotropic motion and discrete conformational substates, often of functional importance. In protein structure determination by X-ray crystallography, the observed diffraction pattern results from the scattering of X-rays by an ensemble of heterogeneous molecules, ordered and oriented by packing in a crystal lattice. The majority of proteins diffract to resolutions where heterogeneity is difficult to identify and model, and are therefore approximated by a single, average conformation with isotropic variance. Here we show that disregarding structural heterogeneity introduces degeneracy into the structure determination process, as many single, isotropic models exist that explain the diffraction data equally well. The large differences among these models imply that the accuracy of crystallographic structures has been widely overestimated. Further, it suggests that analyses that depend on small differences in the relative positions of atoms may be flawed. 相似文献
19.
The addition of phenols to hexameric insulin solutions produces a particularly stable hexamer, resulting from a rearrangement in which residues B1-B8 change from an extended conformation (T-state) to form an alpha-helix (R-state). The R-state is, in part, stabilized by nonpolar interactions between the phenolic molecule and residue B5 His at the dimer-dimer interface. The B5 His --> Tyr mutant human insulin was constructed to see if the tyrosine side chain would mimic the effect of phenol binding in the hexamer and induce the R-state. In partial support of this hypothesis, the molecule crystallized as a half-helical hexamer (T(3)R(3)) in conditions that conventionally promote the fully nonhelical (T6) form. As expected, in the presence of phenol or resorcinol, the B5 Tyr hexamers adopt the fully helical (R6) conformation. Molecular modeling calculations were performed to investigate the conformational preference of the T-state B5 Tyr side chain in the T(3)R(3) form, this side chain being associated with structural perturbations of the A7-A10 loop in an adjacent hexamer. For an isolated dimer, several different orientations of the side chain were found, which were close in energy and readily interconvertible. In the crystal environment only one of these conformations remains low in energy; this conformation corresponds to that observed in the crystal structure. This suggests that packing constraints around residue B5 Tyr result in the observed structural rearrangements. Thus, rather than promoting the R-state in a manner analogous to phenol, the mutation appears to destabilize the T-state. These studies highlight the role of B5 His in determining hexamer conformation and in mediating crystal packing interactions, properties that are likely be important in vivo. 相似文献
20.
Nicholas Furnham Paul IW de Bakker Swanand Gore David F Burke Tom L Blundell 《BMC structural biology》2008,8(1):7