Effect of thermoinduced changes in T4 bacteriophage structure on the process of molecular recognition of 'host' cells.

By means of high-precision acoustic measurements and by methods of fluorescent and electron microscopy, investigations have been performed of thermoinduced conformational changes in T4 bacteriophage and its thermolabile mutants altered in baseplate proteins (gene products 7, 8, 10). A relationship was found between the conformational changes in T4 bacteriophage structure in the temperature range of 33-45 degrees C and the efficiency of bacteriophage adsorption and the changes in the orientation of long tail fibers. The possibility of heat regulation of 'recognition' of 'host' cells by bacterial viruses is suggested.     

Molecular assembly and structure of the bacteriophage T4 tail

The tail of bacteriophage T4 undergoes large structural changes upon infection while delivering the phage genome into the host cell. The baseplate is located at the distal end of the contractile tail and plays a central role in transmitting the signal to the tail sheath that the tailfibers have been adsorbed by a host bacterium. This then triggers the sheath contraction. In order to understand the mechanism of assembly and conformational changes of the baseplate upon infection, we have determined the structure of an in vitro assembled baseplate through the three-dimensional reconstruction of cryo-electron microscopy images to a resolution of 3.8 Å from electron micrographs. The atomic structure was fitted to the baseplate structure before and after sheath contraction in order to elucidate the conformational changes that occur after bacteriophage T4 has attached itself to a cell surface. The structure was also used to investigate the protease digestion of the assembly intermediates and the mutation sites of the tail genes, resulting in a number of phenotypes.     

Movement and self-control in protein assemblies. Quasi-equivalence revisited.

Purposeful switching among different conformational states exerts self-control in the construction and action of protein assemblies. Quasi-equivalence, conceived to explain icosahedral virus structure, arises by differentiation of identical protein subunits into different conformations that conserve essential bonding specificity. Mechanical models designed to represent the energy distribution in the structure, rather than just the arrangement of matter, are used to explore flexibility and self-controlled movements in virus particles. Information about the assembly of bacterial flagella, actin, tobacco mosaic virus and the T4 bacteriophage tail structure show that assembly can be controlled by switching the subunits from an inactive, unsociable form to an active, associable form. Energy to drive this change is provided by the intersubunit bonding in the growing structure; this self-control of assembly by conformational switching is called "autostery", by homology with allostery. A mechanical model of the contractile T4 tail sheath has been constructed to demonstrate how self-controlled activation of a latent bonding potential can drive a purposeful movement. The gradient of quasi-equivalent conformations modelled in the contracting tail sheath has suggested a workable mechanism for self-determination of tail tube length. Concerted action by assemblies of identical proteins may often depend on individually differentiated movements.     

Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides

We have determined the crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7. The structure reveals how subunit contacts stabilize the hexamer. Deviation from expected six-fold symmetry of the hexamer indicates that the structure is of an intermediate on the catalytic pathway. The structural consequences of the asymmetry suggest a "binding change" mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding. The structure of a complex with a nonhydrolyzable ATP analog provides additional evidence for this hypothesis, with only four of the six possible nucleotide binding sites being occupied in this conformation of the hexamer. This model suggests a mechanism for DNA translocation.     

Signal transduction at a protein synapse

Contraction of the bacteriophage T4 tail in the act of host cell penetration represents a massive structural change powered by conformational free energy. A paper in this issue of Cell by compares cryo-electron microscopic reconstructions of the initial and final states and reveals that the basic underlying mechanism is concerted rigid-body movements of the constituent protein subunits, akin to the tumbling of gears in a lock.     

Control of virus assembly: HK97 "Whiffleball" mutant capsids without pentons

The capsid of Escherichia coli bacteriophage HK97 assembles as a 420 subunit icosahedral shell called Prohead I which undergoes a series of maturation steps, including proteolytic cleavage, conformational rearrangements, and covalent cross-linking among all the subunits to yield the highly stable mature Head II shell. Prohead I have been shown to assemble from pre-formed hexamers and pentamers of the capsid protein subunit. We report here the properties of a mutant of the capsid protein, E219K, which illuminate the assembly of Prohead I. The mutant capsid protein is capable of going through all of the biochemically and morphologically defined steps of capsid maturation, and when it is expressed by itself from a plasmid it assembles efficiently into a Prohead I that is morphologically indistinguishable from the wild-type Prohead I, with a full complement of both hexamers and pentamers. Unlike the wild-type Prohead I, when the mutant structure is dissociated into capsomers in vitro, only hexamers are found. When such preparations are put under assembly conditions, these mutant hexamers assemble into "Whiffleballs", particles that are identical with Prohead I except that they are missing the 12 pentamers. These Whiffleballs can even be converted to Prohead I by specifically binding wild-type pentamers. We argue that the ability of the mutant hexamers to assemble in the absence of pentamers implies that they retain a memory of their earlier assembled state, most likely as a conformational difference relative to assembly-naive hexamers. The data therefore favor a model in which Prohead I assembly is regulated by conformational switching of the hexamer.     

Functional and structural roles of critical amino acids within the"N", "P", and "A" domains of the Ca2+ ATPase (SERCA) headpiece

Twenty five amino acids within the "N", "P", and "A" domains of the Ca(2+) ATPase (SERCA1) headpiece were subjected to site directed mutagenesis, taking advantage of a high yield expression system. Functional and conformational effects of mutations were interpreted systematically in the light of the high resolution WT structure, defining direct involvement in catalysis as well as in stabilization of various positions acquired by each domain upon substrate binding and utilization. Amino acids involved in binding of ATP (such as Phe487 and Arg560 in the N domain) or phosphate (such as Asp351, Thr625, Lys684, and Thr353 in the P domain) were characterized with respect to their binding mechanism. Further identified were "positional" roles of several amino acids that stabilize neighboring residues for optimal binding of substrate or Mg(2+), or interface between headpiece domains as they change their relative positions in the course of the catalytic cycle. These include cross-linking of the "N" and "P" domains (e.g., Arg560/Asp627 salt bridge to stabilize domain approximation by ATP binding), and stabilization of the "A", "N", and activated "P" domains in arrangements differing from the ground E2 state and driven by catalytic events. This stabilization is produced through hydrogen bonds at domain interfaces, which vary depending on the intermediate state (e.g., Glu486/T171 in E1P and E2P, as opposed to Glu486/H190 in E2). We demonstrate that specific arrangements of the headpiece domains shown in crystal structures are, in fact, required to trigger displacement of transmembrane segments during the enzyme cycle in solution, allowing long range linkage of catalytic and Ca(2+) binding functions.     

DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process. Nucleotide hydrolysis is not required

Many helicases assemble into ring-shaped hexamers and bind DNA in their central channel. This raises the question as to how the DNA gets into the central channel to form a topologically linked complex. We have used the presteady-state stopped-flow kinetic method and protein fluorescence changes to investigate the mechanism of single-stranded DNA (ssDNA) binding to the bacteriophage T7 helicase-primase, gp4A'. We have found that the kinetics of 30-mer ssDNA binding to a preformed gp4A' hexamer in the presence of both Mg-dTMP-PCP and Mg-dTTP are similar, indicating that Mg-dTTP binding is sufficient and hydrolysis is not necessary for efficient DNA binding. Multiple transient changes in gp4A' fluorescence revealed a four-step mechanism for DNA binding with Mg-dTTP. These transient changes were analyzed by global fitting and kinetic simulation to determine the intrinsic rate constants of this four-step mechanism. The initial steps, including the bimolecular encounter of the DNA with the helicase and a subsequent conformational change, were fast. We propose that these initial steps of DNA binding occur at a readily accessible site, which is likely to be on the outside of the hexamer ring. The binding of the 30-mer ssDNA at this loading site is followed by slower conformational changes that allow the DNA to transit into the central channel of gp4A' via a ring-opening or threading pathway.     

A simulation of T4 bacteriophage assembly and operation

A model is presented for the self-assembly and operation of a bacteriophage comparable with the T4 bacteriophage that infects Escherichia coli. The model treats protein molecules as simple units obeying the principle free energy minimization, and exhibiting the properties of quasi-equivalence and conformational switching. A computer program incorporating the model has been developed. The results of simulation using this program are presented.     

Conformational rearrangements of bacteriophage T4 lysozyme during its binding to the inhibitor]

The structural changes of bacteriophage T4 lysozyme during its binding to the inhibitor, i. e. disaccharide-tetrapeptide N-acetylglucosaminyl-N-acetylmuraminyl - L - alanyl-gamma-D-glutaminyl - mesodiaminopimelyl-D-alanine) isolated from Escherichia coli cell wall have been studied. During the inhibitor binding to the protein the degree of helicity decreases by approximately 14% as was shown using the circular dichroism technique. The changes in optical properties of tryptophane, tyrosine and phenylalanine residues detected by UV difference and fluorescence spectroscopy have been observed. Based on the experimental data and a comparison of spatial organization of phage T4 lysozyme and chicken egg-white lysozyme made it possible to develop a structural model of phage T4 lysozyme functioning. This model may account for the differences in specificity of action of bacteriophage T4 and chicken egg-white lysozymes and allows to establish the role of the "extra" part of phage lysozyme. According to the model, at the first stage of binding the peptide part of the substrate comes in contact with the "upper" (with respect to the cleft) part of the protein molecule (residues 106--116 and 135--140). This results in rearrangement of the molecule, with opening of the cleft at the second stage. This makes possible the access of the polysaccharide part of the substrate of the active site and a subsequent hydrolysis of the beta (1 leads to 4) glycoside bond.     

Optimized incorporation of an unnatural fluorescent amino acid affords measurement of conformational dynamics governing high-fidelity DNA replication

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli. MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication.     

Reconciling the "old" and "new" views of protein allostery: a molecular simulation study of chemotaxis Y protein (CheY)

A combination of thirty-two 10-ns-scale molecular dynamics simulations were used to explore the coupling between conformational transition and phosphorylation in the bacteria chemotaxis Y protein (CheY), as a simple but representative example of protein allostery. Results from these simulations support an activation mechanism in which the beta4-alpha4 loop, at least partially, gates the isomerization of Tyr106. The roles of phosphorylation and the conserved Thr87 are deemed indirect in that they stabilize the active configuration of the beta4-alpha4 loop. The indirect role of the activation event (phosphorylation) and/or conserved residues in stabilizing, rather than causing, specific conformational transition is likely a feature in many signaling systems. The current analysis of CheY also helps to make clear that neither the "old" (induced fit) nor the "new" (population shift) views for protein allostery are complete, because they emphasize the kinetic (mechanistic) and thermodynamic aspects of allosteric transitions, respectively. In this regard, an issue that warrants further analysis concerns the interplay of concerted collective motion and sequential local structural changes in modulating cooperativity between distant sites in biomolecules.     

The maturation-dependent conformational change of the major capsid protein of bacteriophage T4 involves a substantial change in secondary structure

We have investigated the conformational basis of the expansion transformation that occurs upon maturation of the bacteriophage T4 prohead, by using laser Raman spectroscopy to determine the secondary structure of the major capsid protein in both the precursor and the mature states of the surface lattice. This transformation involves major changes in the physical, chemical, and immunological properties of the capsid and is preceded in vivo by processing of its major protein, gp23 (56 kDa), to gp23* (49 kDa), by proteolysis of its N-terminal gp23-delta domain. The respective secondary structures of gp23 in the unexpanded state, and of gp23* in the expanded state, were determined from the laser Raman spectra of polyheads, tubular polymorphic variants of the capsid. Similar measurements were also made on uncleaved polyheads that had been expanded in vitro and, for reference, on thermally denatured polyheads. We find that, with or without cleavage of gp23, expansion is accompanied by substantial changes in secondary structure, involving a major reduction in alpha-helix content and an increase in beta-sheet. The beta-sheet contents of gp23* or gp23 in the expanded state of the surface lattice, and even of gp23 in the unexpanded state, are sufficient for a domain with the "jellyroll" fold of antiparallel beta-sheets, previously detected in the capsid proteins of other icosahedral viruses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synonymous codon preferences in bacteriophage T4: a distinctive use of transfer RNAs from T4 and from its host Escherichia coli.

Codon usage data of bacteriophage T4 genes were compiled and synonymous codon preferences were investigated in comparison with tRNA availabilities in an infected cell. Since the genome of T4 is highly AT rich and its codon usage pattern is significantly different from that of its host Escherichia coli, certain codons of T4 genes need to be translated by appropriate host transfer RNAs present in minor amounts. To avoid this predicament, T4 phage seems to direct the synthesis of its own tRNA molecules and these phage tRNAs are suggested to supplement the host tRNA population with isoacceptors that are normally present in minor amounts. A positive correlation was found in that the frequency of E. coli optimal codons in T4 genes increases as the number of protein monomers per phage particle increases. A negative correlation was also found between the number of protein monomers per phage and the frequency of "T4 optimal codons", which are defined as those codons that are efficiently recognized by T4 tRNAs. From these observations it was proposed that tRNAs from the host are predominantly used for translation of highly expressed T4 genes while tRNAs from T4 tend to be used for translation of weakly expressed T4 genes. This distinctive tRNA-usage in T4 may be an optimization of translational efficiency, and an adjustment of T4-encoded tRNAs to the synonymous codon preferences, which are largely influenced by the high genomic AT-content, would have occurred during evolution.     

A conformational switch in bacteriophage p22 portal protein primes genome injection

Double-stranded DNA (dsDNA) viruses such as herpesviruses and bacteriophages infect by delivering their genetic material into cells, a task mediated by a DNA channel called "portal protein." We have used electron cryomicroscopy to determine the structure of bacteriophage P22 portal protein in both the procapsid and mature capsid conformations. We find that, just as the viral capsid undergoes major conformational changes during virus maturation, the portal protein switches conformation from a procapsid to a mature phage state upon binding of gp4, the factor that initiates tail assembly. This dramatic conformational change traverses the entire length of the DNA channel, from the outside of the virus to the inner shell, and erects a large dome domain directly above the DNA channel that binds dsDNA inside the capsid. We hypothesize that this conformational change primes dsDNA for injection and directly couples completion of virus morphogenesis to a new cycle of infection.     

Raman spectroscopy of the "potential sensor" of potential-dependent channels.

Raman spectroscopy (RS) study shows that the "potential sensor" responds to changes in intramembrane potential by conformational changes. The mechanism of regulation of the channel "gate" by the carotenoid potential sensor is discussed.     

Conformational analysis corresponding to intra-chain disulfide bridged peptides in proteins of known three-dimensional structure

We have carried out a systematic analysis in order to evaluate whether Intra-Chain Disulfide Bridged Peptides (ICDBPs) observed in proteins of known three-dimensional structure adopt structurally similar conformations as they may correspond to structural/functional motifs. 406 representative ICDBPs comprising between 3 to 17 amino acid residues could be classified according to peptide sequence length and main-chain secondary structure conformation into 146 classes. ICDBPs comprising 6 amino acid residues are maximally represented in the Protein Data Bank. They also represent the maximum number of main-chain secondary structure conformational classes. Individual ICDBPs in each class represent different protein superfamilies and correspond to different amino acid sequences. We identified 145 ICDBP pairs that had not less-than 0.5 A root mean square deviation value corresponding to their equivalent peptide backbone atoms. We believe these ICDBPs represent structural motifs and possible candidates in order to further explore their structure/function role in the corresponding proteins. The common conformational classes observed for ICDBPs defined according to the main-chain secondary structure conformations; H (helix), B (residue in a isolated beta bridge), C (coil), E (extended beta strand), G (3(10) helix), I (pi helix), S (bend), T (hydrogen-bonded turn) were; "CHHH", "CTTC", "CSSS" and "CSSC" (for ICDBP length 4), "CSSCC" (length 5), "EETTEE", "CCSSCC", "CCSSSC" (length 6), "EETTTEE" (length 7), "EETTTTEE" (length 8), "EEEETTEEEE" (length 10), "EEEETTTEEEE" (length 11) and "EEEETTTTEEEE" (length 12).