Biomedical subjects

Marc C Morais

Publications and source records attributed to Marc C Morais.

10 recordsLinked to original sources

PubMed2006

From structure of the complex to understanding of the biology.

The most extensive structural information on viruses relates to apparently icosahedral virions and is based on X-ray crystallography and on cryo-electron microscopy (cryo-EM) single-particle reconstructions. Both techniques lean heavily on imposing icosahedral symmetry, thereby obscuring any deviation from the assumed symmetry. However, tailed bacteriophages have icosahedral or prolate icosahedral heads that have one obvious unique vertex where the genome can enter for DNA packaging and exit when infecting a host cell. The presence of the tail allows cryo-EM reconstructions in which the special vertex is used to orient the head in a unique manner. Some very large dsDNA icosahedral viruses also develop special vertices thought to be required for infecting host cells. Similarly, preliminary cryo-EM data for the small ssDNA canine parvovirus complexed with receptor suggests that these viruses, previously considered to be accurately icosahedral, might have some asymmetric properties that generate one preferred receptor-binding site on the viral surface. Comparisons are made between rhinoviruses that bind receptor molecules uniformly to all 60 equivalent binding sites, canine parvovirus, which appears to have a preferred receptor-binding site, and bacteriophage T4, which gains major biological advantages on account of its unique vertex and tail organelle.

Bacteriophages↗

PubMed2006

Molecular dissection of ø29 scaffolding protein function in an in vitro assembly system.

An in vitro assembly system was developed to study prolate capsid assembly of phage ø29 biochemically, and to identify regions of scaffolding protein required for its functions. The crowding agent polyethylene glycol can induce bacteriophage ø29 monomeric capsid protein and dimeric scaffolding protein to co-assemble to form particles which have the same geometry as either prolate T=3 Q=5 procapsids formed in vivo or previously observed isometric particles. The formation of particles is a scaffolding-dependent reaction. The balance between the fidelity and efficiency of assembly is controlled by the concentration of crowding agent and temperature. The assembly process is salt sensitive, suggesting that the interactions between the scaffolding and coat proteins are electrostatic. Three N-terminal ø29 scaffolding protein deletion mutants, Delta 1-9, Delta 1-15 and Delta 1-22, abolish the assembly activity. Circular dichroism spectra indicate that these N-terminal deletions are accompanied by a loss of helicity. The inability of these proteins to dimerize suggests that the N-terminal region of the scaffolding protein contributes to the dimer interface and maintains the structural integrity of the dimeric protein. Two C-terminal scaffolding protein deletion mutants, Delta 79-97 and Delta 62-97, also fail to promote assembly. However, the secondary structure and the dimerization ability of these mutants are unchanged relative to wild-type, which suggests that the C terminus is the likely site of interaction with the capsid protein.

Bacteriophage P22↗

PubMed2006

Structural changes of bacteriophage phi29 upon DNA packaging and release.

Cryo-electron microscopy three-dimensional reconstructions have been made of mature and of emptied bacteriophage phi29 particles without making symmetry assumptions. Comparisons of these structures with each other and with the phi29 prohead indicate how conformational changes might initiate successive steps of assembly and infection. The 12 adsorption capable 'appendages' were found to have a structure homologous to the bacteriophage P22 tailspikes. Two of the appendages are extended radially outwards, away from the long axis of the virus, whereas the others are around and parallel to the phage axis. The appendage orientations are correlated with the symmetry-mismatched positions of the five-fold related head fibers, suggesting a mechanism for partial cell wall digestion upon rotation of the head about the tail when initiating infection. The narrow end of the head-tail connector is expanded in the mature virus. Gene product 3, bound to the 5' ends of the genome, appears to be positioned within the expanded connector, which may potentiate the release of DNA-packaging machine components, creating a binding site for attachment of the tail.

Bacillus Phages↗

PubMed2006

Determinants of bacteriophage phi29 head morphology.

Bacteriophage phi29 requires scaffolding protein to assemble the 450 x 540 A prolate prohead with T = 3 symmetry end caps. In infections with a temperature-sensitive mutant scaffolding protein, capsids assemble predominantly into 370 A diameter isometric particles with T = 3 symmetry that lack a head-tail connector. However, a few larger, 430 A diameter, particles are also assembled. Cryo-electron microscopy shows that these larger particles are icosahedral with T = 4 symmetry. The prolate prohead, as well as the two isometric capsids with T = 3 and T = 4 symmetry, are composed of similar pentamers and differently skewed hexamers. The skewing of the hexamers in the equatorial region of proheads and in the T = 4 isometric particles reflects their different environments. One of the functions of the scaffolding protein, present in the prohead, may be to stabilize skewed hexamers during assembly.

Bacillus subtilis↗

PubMed2005

Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29.

Bacteriophage phi29 is one of the smallest and simplest known dsDNA phages, making it amenable to structural investigations. The three-dimensional structure of a fiberless, isometric variant has been determined to 7.9 A resolution by cryo-electron microscopy (cryo-EM), allowing the identification of alpha helices and beta sheets. Their arrangement indicates that the folds of the phi29 and bacteriophage HK97 capsid proteins are similar except for an additional immunoglobulin-like domain of the phi29 protein. An atomic model that incorporates these two domains fits well into the cryo-EM density of the T = 3, fiberless isometric phi29 particle, and cryo-EM structures of fibered isometric and fiberless prolate prohead phi29 particles at resolutions of 8.7 A and 12.7 A, respectively. Thus, phi29 joins the growing number of phages that utilize the HK97 capsid structure, suggesting that this protein fold may be as prevalent in capsids of dsDNA phages as the jelly roll fold is in eukaryotic viruses.

Bacillus Phages↗

PubMed2005

Combining X-ray crystallography and electron microscopy.

The combination of cryo-electron microscopy to study large biological assemblies at low resolution with crystallography to determine near atomic structures of assembly fragments is quickly expanding the horizon of structural biology. This technique can be used to advantage in the study of large structures that cannot be crystallized, to follow dynamic processes, and to "purify" samples by visual selection of particles. Factors affecting the quality of cryo-electron microscopy maps and limits of accuracy in fitting known structural fragments are discussed.

Cryoelectron Microscopy↗

PubMed2004

Conformational switching by the scaffolding protein D directs the assembly of bacteriophage phiX174.

The three-dimensional structure of bacteriophage phiX174 external scaffolding protein D, prior to its interaction with other structural proteins, has been determined to 3.3 angstroms by X-ray crystallography. The crystals belong to space group P4(1)2(1)2 with a dimer in the asymmetric unit that closely resembles asymmetric dimers observed in the phiX174 procapsid structure. Furthermore, application of the crystallographic 4(1) symmetry operation to one of these dimers generates a tetramer similar to the tetramer in the icosahedral asymmetric unit of the procapsid. These data suggest that both dimers and tetramers of the D protein are true morphogenetic intermediates and can form independently of other proteins involved in procapsid morphogenesis. The crystal structure of the D scaffolding protein thus represents the state of the polypeptide prior to procapsid assembly. Hence, comparison with the procapsid structure provides a rare opportunity to follow the conformational switching events necessary for the construction of complex macromolecular assemblies.

Bacteriophage phi X 174↗

PubMed2004

Investigation of metal ion binding in phosphonoacetaldehyde hydrolase identifies sequence markers for metal-activated enzymes of the HAD enzyme superfamily.

The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an alpha,beta-core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.

Alanine↗

PubMed2003

X-ray crystallographic and site-directed mutagenesis analysis of the mechanism of Schiff-base formation in phosphonoacetaldehyde hydrolase catalysis.

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) to form orthophosphate and acetaldehyde. The reaction proceeds via a Schiff-base intermediate formed between Lys-53 and the Pald carbonyl. The x-ray crystal structures of the wild-type phosphonatase complexed with Mg(II) alone or with Mg(II) plus vinylsulfonate (a phosphonoethylenamine analog) were determined to 2.8 and 2.4 A, respectively. These structures were used to determine the identity and positions of active site residues surrounding the Lys-53 ammonium group and the Pald carbonyl. These include Cys-22, His-56, Tyr-128, and Met-49. Site-directed mutagenesis was then employed to determine whether or not these groups participate in catalysis. Based on rate contributions, Tyr-128 and Cys-22 were eliminated as potential catalytic groups. The Lys-53 epsilon-amino group, positioned for reaction with the Pald carbonyl, forms a hydrogen bond with water 120. Water 120 is also within hydrogen bond distance of an imidazole nitrogen of His-56 and the sulfur atom of Met-49. Kinetic constants for mutants indicated that His-56 (1000-fold reduction in k(cat)/K(m) upon Ala substitution) and Met-49 (17,000-fold reduction in k(cat)/K(m) upon Leu substitution) function in catalysis of Schiff-base formation. Based on these results, it is proposed that a network of hydrogen bonds among Lys-53, water 120, His-56, and Met-49 facilitate proton transfer from Lys-53 to the carbinolamine intermediate. Comparison of the vinylsulfonate complex versus unliganded structures indicated that association of the cap and core domains is essential for the positioning of the Lys-53 for attack at the Pald carbonyl and that substrate binding at the core domain stabilizes cap domain binding.

Acetaldehyde↗

PubMed2003

Bacteriophage phi29 scaffolding protein gp7 before and after prohead assembly.

Three-dimensional structures of the double-stranded DNA bacteriophage phi29 scaffolding protein (gp7) before and after prohead assembly have been determined at resolutions of 2.2 and 2.8 A, respectively. Both structures are dimers that resemble arrows, with a four-helix bundle composing the arrowhead and a coiled coil forming the tail. The structural resemblance of gp7 to the yeast transcription factor GCN4 suggests a DNA-binding function that was confirmed by native gel electrophoresis. DNA binding to gp7 may have a role in mediating the structural transition from prohead to mature virus and scaffold release. A cryo-EM analysis indicates that gp7 is arranged inside the capsid as a series of concentric shells. The position of the higher density features in these shells correlates with the positions of hexamers in the equatorial region of the capsid, suggesting that gp7 may regulate formation of the prolate head through interactions with these hexamers.

Bacillus Phages↗