A Molecular Staple: D-Loops in the I Domain of Bacteriophage P22 Coat Protein Make Important Intercapsomer Contacts Required for Procapsid Assembly

ABSTRACTBacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as “molecular staples” at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging.IMPORTANCEMany dsDNA viruses have morphogenic pathways utilizing an intermediate capsid, known as a procapsid. These procapsids are assembled from a coat protein having the HK97 fold in a reaction driven by scaffolding proteins or delta domains. Maturation of the capsid occurs during DNA packaging. Bacteriophage HK97 uniquely stabilizes its capsid during maturation by intercapsomer cross-linking, but most virus capsids are stabilized by alternate means. Here we show that the I domain that is inserted into the coat protein of bacteriophage P22 is important in the process of proper procapsid assembly. Specifically, the I domain allows for stabilizing interactions across the capsid 2-fold axis of symmetry via a D-loop. When amino acid residues at the tip of the D-loop are mutated, aberrant assembly products, including tubes, are formed instead of procapsids, consequently phage production is affected, indicating the importance of stabilizing interactions during the assembly and maturation reactions.

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Statistical Force-Field for Structural Modeling Using Chemical Cross-Linking/mass Spectrometry Distance Constraints

10.26434/chemrxiv.6030563 ◽

2018 ◽

Author(s):

Allan J. R. Ferrari ◽

Fabio C. Gozzo ◽

Leandro Martinez

Keyword(s):

Mass Spectrometry ◽

Amino Acid ◽

Force Field ◽

Protein Structures ◽

Protein Structure Determination ◽

Structural Modeling ◽

Cross Linking ◽

Amino Acid Residues ◽

Distance Constraints ◽

Chemical Cross Linking

<div><p>Chemical cross-linking/Mass Spectrometry (XLMS) is an experimental method to obtain distance constraints between amino acid residues, which can be applied to structural modeling of tertiary and quaternary biomolecular structures. These constraints provide, in principle, only upper limits to the distance between amino acid residues along the surface of the biomolecule. In practice, attempts to use of XLMS constraints for tertiary protein structure determination have not been widely successful. This indicates the need of specifically designed strategies for the representation of these constraints within modeling algorithms. Here, a force-field designed to represent XLMS-derived constraints is proposed. The potential energy functions are obtained by computing, in the database of known protein structures, the probability of satisfaction of a topological cross-linking distance as a function of the Euclidean distance between amino acid residues. The force-field can be easily incorporated into current modeling methods and software. In this work, the force-field was implemented within the Rosetta ab initio relax protocol. We show a significant improvement in the quality of the models obtained relative to current strategies for constraint representation. This force-field contributes to the long-desired goal of obtaining the tertiary structures of proteins using XLMS data. Force-field parameters and usage instructions are freely available at http://m3g.iqm.unicamp.br/topolink/xlff <br></p></div><p></p><p></p>

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Modulation of lysyl oxidase activity toward peptidyl lysine by vicinal dicarboxylic amino acid residues. Implications for collagen cross-linking.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)31798-2 ◽

1994 ◽

Vol 269 (35) ◽

pp. 22366-22371 ◽

Cited By ~ 1

Author(s):

N. Nagan ◽

H.M. Kagan

Keyword(s):

Amino Acid ◽

Oxidase Activity ◽

Lysyl Oxidase ◽

Cross Linking ◽

Amino Acid Residues ◽

Dicarboxylic Amino Acid ◽

Collagen Cross Linking

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Cysteine Residues in the Transmembrane Regions of M13 Procoat Protein Suggest that Oligomeric Coat Proteins Assemble onto Phage Progeny

Journal of Bacteriology ◽

10.1128/jb.01551-06 ◽

2007 ◽

Vol 189 (7) ◽

pp. 2897-2905 ◽

Cited By ~ 15

Author(s):

Christof Nagler ◽

Gisela Nagler ◽

Andreas Kuhn

Keyword(s):

Coat Protein ◽

Leader Peptide ◽

Cysteine Residues ◽

Coat Proteins ◽

Amino Acid Residues ◽

Major Coat Protein ◽

Phage Dna ◽

Phage Assembly ◽

Transmembrane Regions ◽

Leader Peptidase

ABSTRACT The M13 phage assembles in the inner membrane of Escherichia coli. During maturation, about 2,700 copies of the major coat protein move from the membrane onto a single-stranded phage DNA molecule that extrudes out of the cell. The major coat protein is synthesized as a precursor, termed procoat protein, and inserts into the membrane via a Sec-independent pathway. It is processed by a leader peptidase from its leader (signal) peptide before it is assembled onto the phage DNA. The transmembrane regions of the procoat protein play an important role in all these processes. Using cysteine mutants with mutations in the transmembrane regions of the procoat and coat proteins, we investigated which of the residues are involved in multimer formation, interaction with the leader peptidase, and formation of M13 progeny particles. We found that most single cysteine residues do not interfere with the membrane insertion, processing, and assembly of the phage. Treatment of the cells with copper phenanthroline showed that the cysteine residues were readily engaged in dimer and multimer formation. This suggests that the coat proteins assemble into multimers before they proceed onto the nascent phage particles. In addition, we found that when a cysteine is located in the leader peptide at the −6 position, processing of the mutant procoat protein and of other exported proteins is affected. This inhibition of the leader peptidase results in death of the cell and shows that there are distinct amino acid residues in the M13 procoat protein involved at specific steps of the phage assembly process.

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Transmembrane signaling in the sensor kinase DcuS ofEscherichia coli: A long-range piston-type displacement of transmembrane helix 2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507217112 ◽

2015 ◽

Vol 112 (35) ◽

pp. 11042-11047 ◽

Cited By ~ 15

Author(s):

Christian Monzel ◽

Gottfried Unden

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Transmembrane Helix ◽

Binding Domain ◽

Cross Linking ◽

Sensor Kinase ◽

Amino Acid Residues ◽

Terminal Part ◽

Type Shift

The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane–water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21–41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states. In contrast, TM2 was inserted with residues 181–201 in the OFF state and residues 185–205 in the ON state. Replacement of Trp 185 by an Arg residue caused displacement of TM2 toward the outside of the membrane and a concomitant induction of the ON state. Results from Cys cross-linking of TM2/TM2′ in the DcuS homodimer excluded rotation; thus, data from accessibility changes of TM2 upon activation, either by ligand binding or by mutation of TM2, and cross-linking of TM2 and the connected region in the periplasm suggest a piston-type shift of TM2 by four residues to the periplasm upon activation (or fumarate binding). This mode of function is supported by the suggestion from energetic calculations of two preferred positions for TM2 insertion in the membrane. The shift of TM2 by four residues (or 4–6 Å) toward the periplasm upon activation is complementary to the periplasmic displacement of 3–4 Å of the C-terminal part of the periplasmic ligand-binding domain upon ligand occupancy in the citrate-binding domain in the homologous CitA sensor kinase.

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A hydrophobic network: Intersubunit and intercapsomer interactions stabilizing the bacteriophage P22 capsid

10.1101/626804 ◽

2019 ◽

Cited By ~ 1

Author(s):

Kunica Asija ◽

Carolyn M. Teschke

Keyword(s):

Coat Protein ◽

Hydrophobic Interactions ◽

Protein Structures ◽

Antiviral Drug ◽

Coat Proteins ◽

Bacteriophage P22 ◽

Outer Periphery ◽

Phage P22 ◽

P Domain ◽

Capsid Stability

AbstractdsDNA tailed phages and herpesviruses assemble their capsids using coat proteins that have the ubiquitous HK97 fold. Though this fold is common, we do not have a thorough understanding of the different ways viruses adapt it to maintain stability in various environments. The HK97-fold E-loop, which connects adjacent subunits at the outer periphery of capsomers, has been implicated in capsid stability. Here we show that in bacteriophage P22, residue W61 at the tip of the E-loop plays a role in stabilizing procapsids and in maturation. We hypothesize that a hydrophobic pocket is formed by residues I366 and W410 in the P-domain of a neighboring subunit within a capsomer, into which W61 fits like a peg. In addition, W61 likely bridges to residues A91 and L401 in P-domain loops of an adjacent capsomer, thereby linking the entire capsid together with a network of hydrophobic interactions. There is conservation of this hydrophobic network in the distantly related P22-like phages, indicating that this structural feature is likely important for stabilizing this family of phages. Thus, our data shed light on one of the varied elegant mechanisms used in nature to consistently build stable viral genome containers through subtle adaptation of the HK97 fold.IMPORTANCESimilarities in assembly reactions and coat protein structures of the dsDNA tailed phages and herpesviruses make phages ideal models to understand capsid assembly and identify potential targets for antiviral drug discovery. The coat protein E-loops of these viruses are involved in both intra-and intercapsomer interactions. In phage P22, hydrophobic interactions peg the coat protein subunits together within a capsomer, where the E-loop hydrophobic residue W61 of one subunit packs into a pocket of hydrophobic residues I366 and W410 of the adjacent subunit. W61 also makes hydrophobic interactions with A91 and L401 of a subunit in an adjacent capsomer. We show these intra-and intercapsomer hydrophobic interactions form a network crucial to capsid stability and proper assembly.

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Solution structure and amino acid residues of C‐terminal RAGE (ctRAGE) involved in binding to Dia‐1 FH1 and its signaling

The FASEB Journal ◽

10.1096/fasebj.25.1_supplement.121.4 ◽

2011 ◽

Vol 25 (S1) ◽

Author(s):

VIVEK RAI ◽

Andres Maldonado ◽

Shi‐Fang Yan ◽

Ann Marie Schmidt ◽

Alexander Shekhtman

Keyword(s):

Amino Acid ◽

Solution Structure ◽

Amino Acid Residues

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Statistical Force-Field for Structural Modeling Using Chemical Cross-Linking/mass Spectrometry Distance Constraints

10.26434/chemrxiv.6030563.v1 ◽

2018 ◽

Author(s):

Allan J. R. Ferrari ◽

Fabio C. Gozzo ◽

Leandro Martinez

Keyword(s):

Mass Spectrometry ◽

Amino Acid ◽

Force Field ◽

Protein Structures ◽

Protein Structure Determination ◽

Structural Modeling ◽

Cross Linking ◽

Amino Acid Residues ◽

Distance Constraints ◽

Chemical Cross Linking

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A Two-Amino Acid Difference in the Coat Protein of Satellite panicum mosaic virus Isolates Is Responsible for Differential Synergistic Interactions with Panicum mosaic virus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-18-0247-r ◽

2019 ◽

Vol 32 (4) ◽

pp. 479-490 ◽

Cited By ~ 1

Author(s):

R. V. Chowda-Reddy ◽

Nathan Palmer ◽

Serge Edme ◽

Gautam Sarath ◽

Frank Kovacs ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Coat Protein ◽

Mosaic Virus ◽

Severe Disease ◽

Amino Acid Difference ◽

Amino Acid Residues ◽

Genomic Rna ◽

Proso Millet ◽

Synergistic Interactions

Panicum mosaic virus (PMV) (genus Panicovirus, family Tombusviridae) and its molecular parasite, Satellite panicum mosaic virus (SPMV), synergistically interact in coinfected proso and pearl millet (Panicum miliaceum L.) plants resulting in a severe symptom phenotype. In this study, we examined synergistic interactions between the isolates of PMV and SPMV by using PMV-NE, PMV85, SPMV-KS, and SPMV-Type as interacting partner viruses in different combinations. Coinfection of proso millet plants by PMV-NE and SPMV-KS elicited severe mosaic, chlorosis, stunting, and eventual plant death compared with moderate mosaic, chlorotic streaks, and stunting by PMV85 and SPMV-Type. In reciprocal combinations, coinfection of proso millet by either isolate of PMV with SPMV-KS but not with SPMV-Type elicited severe disease synergism, suggesting that SPMV-KS was the main contributor for efficient synergistic interaction with PMV isolates. Coinfection of proso millet plants by either isolate of PMV and SPMV-KS or SPMV-Type caused increased accumulation of coat protein (CP) and genomic RNA copies of PMV, compared with infections by individual PMV isolates. Additionally, CP and genomic RNA copies of SPMV-KS accumulated at substantially higher levels, compared with SMPV-Type in coinfected proso millet plants with either isolate of PMV. Hybrid viruses between SPMV-KS and SPMV-Type revealed that SPMV isolates harboring a CP fragment with four differing amino acids at positions 18, 35, 59, and 98 were responsible for differential synergistic interactions with PMV in proso millet plants. Mutation of amino acid residues at these positions in different combinations in SPMV-KS, similar to those as in SPMV-Type or vice-versa, revealed that A35 and R98 in SPMV-KS CP play critical roles in enhanced synergistic interactions with PMV isolates. Taken together, these data suggest that the two distinct amino acids at positions 35 and 98 in the CP of SPMV-KS and SPMV-Type are involved in the differential synergistic interactions with the helper viruses.

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A structural model for desmosine cross-linked peptides

Biochemical Journal ◽

10.1042/bj1730617 ◽

1978 ◽

Vol 173 (2) ◽

pp. 617-625 ◽

Cited By ~ 28

Author(s):

R P Mecham ◽

J A Foster

Keyword(s):

Amino Acid ◽

Structural Model ◽

Partial Sequence ◽

Cross Linking ◽

Amino Acid Residues ◽

Cross Link ◽

Link Model ◽

The Cross ◽

Precursor Molecules

Desmosine-enriched peptides were isolated from a thermolysin digest of bovine ligamentum nuchae elastin and a partial sequence was determined. A ‘two-cross-link’ model is proposed in which a second cross-link, perhaps lysinonorleucine, joins two peptide chains approx. 35 amino acid residues removed from the desmosine. Implied in this model is a certain asymmetry or directionality which places restrictions on the ‘sense’ of the peptide chains (either always parallel or anti-parallel) in order to align the cross-linking sites. Imposing such restrictions raises the possibility of specific alignment of elastin precursor molecules by microfibrillar proteins and/or aligning peptides on the precursor molecules themselves.

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Properties of the ribonucleic acid bacteriophage ZIK/1 coat protein and its synthesis in an Escherichia coli cell-free system

Biochemical Journal ◽

10.1042/bj1280481 ◽

1972 ◽

Vol 128 (3) ◽

pp. 481-489 ◽

Cited By ~ 1

Author(s):

J. W. Robinson

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Amino Acid ◽

Coat Protein ◽

Major Product ◽

Coli Cell ◽

Coat Proteins ◽

Protein Subunit ◽

Free System ◽

Cell Free System

The coat protein subunit of the RNA bacteriophage ZIK/1 has a molecular weight of 12100 and does not contain histidine, methionine and cysteine. The amino acid composition of the coat protein is different from that of other RNA bacteriophage coat proteins. Bacteriophage ZIK/1 belongs to a class of RNA bacteriophages distinct from the f2 type, which lack histidine in their coat proteins, and the Qβ type, which lack histidine and methionine. Bacteriophage ZIK/1 RNA is an efficient template in the Escherichia coli cell-free system producing coat protein as the major product and a number of non-coat proteins. This result is similar to that obtained with RNA from f2-type bacteriophages. It is probable that the genomes of RNA bacteriophages are structurally similar and that differences between the types of RNA bacteriophage arise from minor differences in RNA sequence.

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