Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro

Prevelige, Peter E.; Thomas, Dennis W.; King, Jonathan

doi:10.1016/0022-2836(88)90555-4

Cited by 180 publications

(268 citation statements)

References 37 publications

Supporting

Mentioning

258

Contrasting

Order By: Relevance

“…4). WT coat protein was assembly-competent and formed procapsids, as seen by the increase in light scattering (31). The tsf coat protein S223F was assembly-incompetent, and F353L had low assembly activity.…”

Section: Resultsmentioning

confidence: 97%

“…Purification of Coat Proteins-WT, S223F, S223F/T166I, F353L, and F353L/T166I used in the following experiments were obtained from empty procapsid shell stocks that were prepared as previously described (31,32,36,37). Briefly, S. typhimurium (DB7155) were grown in LB to 1 ϫ 10 8 cells/ml at 30°C and infected with various strains of bacteriophage P22 at a multiplicity of infection of 0.075 to prepare a large phage stock.…”

Section: Methodsmentioning

confidence: 99%

“…Based on the above observations, we present a model of folding and assembly of coat protein (Fig. 1 (31). The tsf coat proteins assemble with slower kinetics than wild type (WT) 2 coat protein as a result of the tsf folding defect (30) and assembly does not correct the conformational defects of tsf coat proteins (32).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Alleviation of a Defect in Protein Folding by Increasing the Rate of Subunit Assembly

Aramli

Teschke

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Understanding the nature of protein grammar is critical because amino acid substitutions in some proteins cause misfolding and aggregation of the mutant protein resulting in a disease state. Amino acid substitutions in phage P22 coat protein, known as tsf (temperature-sensitive folding) mutations, cause folding defects that result in aggregation at high temperatures. We have isolated global su (suppressor) amino acid substitutions that alleviate the tsf phenotype in coat protein (Aramli, L. A., and Teschke, C. M. (1999) J. Biol. Chem. 274, 22217-22224). Unexpectedly, we found that a global su amino acid substitution in tsf coat proteins made aggregation worse and that the tsf phenotype was suppressed by increasing the rate of subunit assembly, thereby decreasing the concentration of aggregation-prone folding intermediates.The primary amino acid sequence of a polypeptide encodes all of the information necessary for folding and assembly pathways, as well as the native three-dimensional structure (2). Substitutions and deletions in the amino acid sequence of a protein can have a significant impact on the ability of a protein to fold or assemble properly. Depending on the protein, such changes in the amino acid sequence can lead to protein misfolding, mislocalization caused by misfolding, or aggregation (3, 4). Amino acid substitutions in p53 lead to a misfolding problem compromising the function of the protein, resulting in cancer (5). Further, there are the diseases that result from mislocalization caused by protein misfolding. For example, in ␣ 1 -antitrypsin deficiency, a single amino acid substitution results in the misfolding of ␣ 1 -antitrypsin, leading to the accumulation of long chain polymers within the hepatocyte. This leads to a reduction of the plasma concentrations of ␣-antitrypsin and predisposes individuals to emphysema and liver disease (6). Therefore, understanding the nature of protein grammar is of paramount interest.Although the process of protein folding is still not completely understood, it is known that larger proteins often have identifiable folding intermediates. These folding intermediates may interact inappropriately before reaching the native state. In fact, protein misfolding is a common problem faced by biotechnology companies that harvest proteins of commercial interest using recombinant DNA technologies in heterologous hosts (7-11). Often these proteins have problems with inclusion body formation, thereby decreasing the yield of pharmaceutically important products. Single amino acid substitutions can affect the folding pathway by shifting the folding from the productive pathway to off-pathway aggregation. For example, the amino acid substitutions in transthyretin causes a shift in the equilibrium between the native state and an aggregation-prone unfolding intermediate, resulting in amyloid formation. Individuals with any of the 50 known amino acid substitutions in transthyretin are predisposed to familial amyloidosis (12-15). During the folding of P22 tailspike proteins with tsf (te...

show abstract

Section: Resultsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Alleviation of a Defect in Protein Folding by Increasing the Rate of Subunit Assembly

Aramli

Teschke

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…P22 procapsids and infectious virions were purified from Salmonella typhimurium using an established procedure (47,51). The samples were applied to Quantifoil grids, flash-frozen using a Vitrobot and imaged.…”

Section: Molecular Mechanism For Capsid Assembly Scaffolding Proteinmentioning

confidence: 99%

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Chen

Baker

Hryc

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

192

317

View full text Add to dashboard Cite

Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8-and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T ¼ 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.sDNA viruses infecting both prokaryotes and eukaryotes share a common assembly pathway proceeding from a precursor (procapsid) to an infectious virion (1-4). In addition to the coat proteins, the procapsid requires scaffolding proteins, absent from the virion, for proper assembly, and a portal for DNA packaging and subsequent DNA ejection. However, despite a half-century of research on icosahedral viruses, it remains unclear how initially identical subunits adopt both hexameric and pentameric conformations in the virus and select the correct locations needed to form closed shells of the proper size (5). Packaging of DNA through the portal is accompanied by the exit of scaffolding proteins from the procapsid and conformational changes in the coat proteins as the capsid matures (2, 6).Understanding the molecular mechanisms of dsDNA virus assembly and maturation requires knowledge of the interactions among the coat, scaffolding, and portal proteins, all of which are essential for these processes. X-ray crystallography (7-9) and electron cryomicroscopy (cryo-EM) (10-12) have yielded nearatomic to atomic resolution models of several dsDNA icosahedral viruses and provided a structural framework of interactions among their coat proteins. However, the structural details of procapsid portal incorporation, scaffolding protein bind...

show abstract

“…For example, coat proteins from other systems form aberrant assemblies in the absence of scaffolding protein, whereas scaffolding proteins alone are relatively inert. When mixed, the scaffolding proteins control fidelity, suppressing the formation of heterogeneous aberrant structures containing only the coat protein (21,33,(42)(43)(44)(45)(46)(47)(48)(49)(50). In contrast, the X174 external scaffolding protein D self-associates to form large heterogeneous spherical complexes (Fig.…”

Section: Discussionmentioning

confidence: 99%

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

Cherwa

Tyson

Bedwell

et al. 2017

J Virol

Self Cite

View full text Add to dashboard Cite

During X174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro. In previous studies, X174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo. We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During X174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.KEYWORDS bacteriophage X174, Microviridae, microvirus, scaffolding protein, virus assembly D ue to their rapid replication cycle, detailed structural information, and well developed genetic and biochemical assays, the microviruses have become an ideal model system for experimental evolution (1-4). The morphogenetic pathway can be characterized under restrictive conditions and adaptive mutations can be mapped onto X-ray structures, providing insights into evolutionary mechanisms and protein structure-function relationships (2-8).

show abstract

Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro

Cited by 180 publications

References 37 publications

Alleviation of a Defect in Protein Folding by Increasing the Rate of Subunit Assembly

Alleviation of a Defect in Protein Folding by Increasing the Rate of Subunit Assembly

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

Contact Info

Product

Resources

About