Critical Salt Bridges Guide Capsid Assembly,Stability, and Maturation Behavior in Bacteriophage HK97

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.     

Ligand-induced conformational changes in the acetylcholine-binding protein analyzed by hydrogen-deuterium exchange mass spectrometry

Recent x-ray crystallographic studies of the acetylcholine-binding protein (AChBP) suggest that loop C, found at the circumference of the pentameric molecule, shows distinctive conformational changes upon antagonist and agonist occupation. We have employed hydrogen-deuterium exchange mass spectrometry to examine the influence of bound ligands on solvent exposure of AChBP. Quantitative measurements of deuterium incorporation are possible for approximately 56% of the Lymnaea AChBP sequence, covering primarily the outer surface of AChBP. In the apoprotein, two regions flanking the ligand occupation site at the subunit interface, loop C (residues 175-193) and loop F (residues 164-171), show greater extents of solvent exchange than other regions of the protein including the N- and C-terminal regions. Occupation by nicotinic agonists, epibatidine and lobeline, and nicotinic antagonists, methyllycaconitine, alpha-bungarotoxin, and alpha-cobratoxin, markedly restricts the exchange of loop C amide protons, influencing both the rates and degrees of exchange. Solvent exposure of loop C and its protection by ligand suggest that in the apoprotein, loop C exhibits rapid fluctuations in an open conformation. Bound agonists restrict solvent exposure through loop closure, whereas the larger antagonists restrict solvent exposure largely through occlusion of solvent. Loop F, found on the complementary subunit surface at the interface, also reveals ligand selective changes in amide proton exchange rates. Agonists do not affect solvent accessibility of loop F, whereas certain antagonists cause subtle accessibility changes. These results reveal dynamic states and fluctuating movements in the vicinity of the binding site for unligated AChBP that can be influenced selectively by ligands.     

Mutations in the fourth EGF-like domain affect thrombomodulin-induced changes in the active site of thrombin

A number of alanine and more conservative mutants of residues in the fourth domain of thrombomodulin (TM) were prepared and assayed for protein C activation and for thrombin binding. Several of the alanine mutations appeared to cause misfolding or structural defects as assessed by poor expression and/or NMR HSQC experiments, while more conservative mutations at the same site appeared to allow correct folding and preserved activity. Several of the conservative mutants bound more weakly to thrombin despite the fact that the fourth domain does not directly contact thrombin in the crystal structure of the thrombin-TM complex. A few of the mutant TM fragments bound thrombin with an affinity similar to that of the wild type but exhibited decreases in k cat for protein C activation. These mutants were also less able to cause a change in the steady state fluorescence of fluorescein-EGR-chloromethylketone bound to the active site of thrombin. These results suggest that some residues within the fourth domain of TM may primarily interact with protein C but others are functionally important for altering the way TM interacts with thrombin. Residues in the fourth domain that primarily affect k cat for protein C activation may do this by changing the active site of thrombin.     

Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation

Phytochelatins (PCs) are glutathione-derived peptides that function in heavy metal detoxification in plants and certain fungi. Recent research in Arabidopsis has shown that PCs undergo long-distance transport between roots and shoots. However, it remains unknown which tissues or vascular systems, xylem or phloem, mediate PC translocation and whether PC transport contributes to physiologically relevant long-distance transport of cadmium (Cd) between shoots and roots. To address these questions, xylem and phloem sap were obtained from Brassica napus to quantitatively analyze which thiol species are present in response to Cd exposure. High levels of PCs were identified in the phloem sap within 24 h of Cd exposure using combined mass spectrometry and fluorescence HPLC analyses. Unexpectedly, the concentration of Cd was more than four-fold higher in phloem sap compared to xylem sap. Cadmium exposure dramatically decreased iron levels in xylem and phloem sap whereas other essential heavy metals such as zinc and manganese remained unchanged. Data suggest that Cd inhibits vascular loading of iron but not nicotianamine. The high ratios [PCs]/[Cd] and [glutathione]/[Cd] in the phloem sap suggest that PCs and glutathione (GSH) can function as long-distance carriers of Cd. In contrast, only traces of PCs were detected in xylem sap. Our results suggest that, in addition to directional xylem Cd transport, the phloem is a major vascular system for long-distance source to sink transport of Cd as PC–Cd and glutathione–Cd complexes.     

The structure, dynamics, and binding of the LA45 module pair of the low-density lipoprotein receptor suggest an important role for LA4 in ligand release

The low-density lipoprotein receptor (LDLR), the primary receptor for cholesterol uptake, binds ligands through its seven LDL-A modules (LAs). We present nuclear magnetic resonance (NMR) and ligand binding measurements on the fourth and fifth modules of the LDLR (LA45), the modules critical for ApoE binding, at physiological pH. Unlike LA5 and all other modules in LDLR, LA4 has a very weak calcium affinity, which probably plays a critical role in endosomal ligand release. The NMR solution structure of each module in the LA45 pair only showed minor differences compared to the analogous domains in previously determined crystal structures. The 12-residue linker connecting the modules, though slightly structured through an interaction with LA4, is highly flexible. Although no intermodule nuclear Overhauser effects were detected, chemical shift perturbations and backbone dynamics suggest cross talk between the two modules. The ligand affinity of both modules is enhanced when the two are linked. LA4 is more flexible than LA5 and remains so even in the module pair, which likely is related to its weaker calcium binding affinity.     

Trypsinogen activation as observed in accelerated molecular dynamics simulations

Serine proteases are involved in many fundamental physiological processes, and control of their activity mainly results from the fact that they are synthetized in an inactive form that becomes active upon cleavage. Three decades ago Martin Karplus's group performed the first molecular dynamics simulations of trypsin, the most studied member of the serine protease family, to address the transition from the zymogen to its active form. Based on the computational power available at the time, only high frequency fluctuations, but not the transition steps, could be observed. By performing accelerated molecular dynamics (aMD) simulations, an interesting approach that increases the configurational sampling of atomistic simulations, we were able to observe the N‐terminal tail insertion, a crucial step of the transition mechanism. Our results also support the hypothesis that the hydrophobic effect is the main force guiding the insertion step, although substantial enthalpic contributions are important in the activation mechanism. As the N‐terminal tail insertion is a conserved step in the activation of serine proteases, these results afford new perspective on the underlying thermodynamics of the transition from the zymogen to the active enzyme.     

Autoantibodies in canine masticatory muscle myositis recognize a novel myosin binding protein-C family member

Inflammatory myopathies are a group of autoimmune diseases that affect muscles. In humans, the most common inflammatory myopathies are polymyositis, dermatomyositis, and inclusion body myositis. Autoantibodies may be found in humans with inflammatory myopathies, and these play an important role in diagnosis and disease classification. However, these Abs are typically not muscle specific. Spontaneously occurring canine inflammatory myopathies may be good parallel disorders and provide insights into human myositis. In dogs with inflammatory myopathy, muscle-specific autoantibodies have been found, especially in masticatory muscle myositis. We have identified the major Ag recognized by the autoantibodies in canine masticatory muscle myositis. This Ag is a novel member of the myosin binding protein-C family, which we call masticatory myosin binding protein-C (mMyBP-C). mMyBP-C is localized not only within the masticatory muscle fibers, but also at or near their cell surface, perhaps making it accessible as an immunogen. The gene for mMyBP-C also exists in humans, and mMyBP-C could potentially play a role in certain human inflammatory myopathies. Understanding the role of mMyBP-C in this canine inflammatory myopathy may advance our knowledge of mechanisms of autoimmune inflammatory muscle diseases, not only in dogs, but also in humans.     

The dynamic structure of thrombin in solution

The backbone dynamics of human α-thrombin inhibited at the active site serine were analyzed using R₁, R₂, and heteronuclear NOE experiments, variable temperature TROSY 2D [¹H-¹⁵N] correlation spectra, and R_ex measurements. The N-terminus of the heavy chain, which is formed upon zymogen activation and inserts into the protein core, is highly ordered, as is much of the double beta-barrel core. Some of the surface loops, by contrast, remain very dynamic with order parameters as low as 0.5 indicating significant motions on the ps-ns timescale. Regions of the protein that were thought to be dynamic in the zymogen and to become rigid upon activation, in particular the γ-loop, the 180s loop, and the Na⁺ binding site have order parameters below 0.8. Significant R_ex was observed in most of the γ-loop, in regions proximal to the light chain, and in the β-sheet core. Accelerated molecular dynamics simulations yielded a molecular ensemble consistent with measured residual dipolar couplings that revealed dynamic motions up to milliseconds. Several regions, including the light chain and two proximal loops, did not appear highly dynamic on the ps-ns timescale, but had significant motions on slower timescales.