Probing conserved helical modules of portal complexes by mass spectrometry-based hydrogen/deuterium exchange - PubMed (original) (raw)

Probing conserved helical modules of portal complexes by mass spectrometry-based hydrogen/deuterium exchange

Sebyung Kang et al. J Mol Biol. 2008.

Abstract

The Double-stranded DNA bacteriophage P22 has a ring-shaped dodecameric complex composed of the 84 kDa portal protein subunit that forms the central channel of the phage DNA packaging motor. The overall morphology of the P22 portal complex is similar to that of the portal complexes of Phi29, SPP1, T3, T7 phages and herpes simplex virus. Secondary structure prediction of P22 portal protein and its threading onto the crystal structure of the Phi29 portal complexes suggested that the P22 portal protein complex shares conserved helical modules that were found in the dodecameric interfaces of the Phi29 portal complex. To identify the amino acids involved in intersubunit contacts in the P22 portal ring complexes and validate the threading model, we performed comparative hydrogen/deuterium exchange analysis of monomeric and in vitro assembled portal proteins of P22 and the dodecameric Phi29 portal. Hydrogen/deuterium exchange experiments provided evidence of intersubunit interactions in the P22 portal complex similar to those in the Phi29 portal that map to the regions predicted to be conserved helical modules.

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Figures

Figure 1

Figure 1

Comparison between the Phi29 and the P22 portal complexes. (A) Overlay of the crystal structure of Phi29 (PDB: 1IJG) onto the P22 virion’s portal density. The Phi29 crystal structure fits well into the electron density of the distal half of the P22 portal complex (B) Putative conserved secondary structure modules in the portal proteins of P22 (residues 240–350) and Phi29 (residues 125–225). The secondary structure of the Phi29 portal complex was obtained from the crystal structure of the Phi29 portal complex (PDB: 1IJG). The secondary structure of the P22 portal protein was predicted using the program PredictProtein (

www.predictprotein.org

). The regions of α-helix (coil) and β-strand (arrow) are indicated and the boundary residues are numbered.

Figure 2

Figure 2

Amino acid sequences of the Phi29 portal protein and hydrogen/deuterium exchange profile for peptide residues 136–159 and 160–185. (A) Amino acid sequence of the Phi29 portal protein. The observed peptic fragments of the Phi29 portal protein are underlined (blue lines). The peptic fragments, which were identified by a combination of exact mass matching and MS/MS sequencing, span ~90% of the protein primary sequence. The secondary structure was determined and mapped as in Figure 1. (B) Mass spectra of the peptic fragment corresponding to residues 136–159 (left panel) and 160–185 (right panel) from the Phi29 portal complexes at various exchange times. In each panel, the bottom spectrum represents the un-exchanged control.

Figure 2

Figure 2

Amino acid sequences of the Phi29 portal protein and hydrogen/deuterium exchange profile for peptide residues 136–159 and 160–185. (A) Amino acid sequence of the Phi29 portal protein. The observed peptic fragments of the Phi29 portal protein are underlined (blue lines). The peptic fragments, which were identified by a combination of exact mass matching and MS/MS sequencing, span ~90% of the protein primary sequence. The secondary structure was determined and mapped as in Figure 1. (B) Mass spectra of the peptic fragment corresponding to residues 136–159 (left panel) and 160–185 (right panel) from the Phi29 portal complexes at various exchange times. In each panel, the bottom spectrum represents the un-exchanged control.

Figure 3

Figure 3

Plots of the number of deuterium atoms incorporated according to the exchange period for peptic fragments from the Phi29 portal complexes (dots). The solid line represents the fit obtained by three component exponential fitting the exchange data (see materials and methods). Peptic fragments (A) 136–159, (B) 197–220, (C) 160–185, (D) 1–13, and (E) 288–308. Highest number on the y-axis represents the theoretical number of exchangeable amide protons.

Figure 3

Figure 3

Plots of the number of deuterium atoms incorporated according to the exchange period for peptic fragments from the Phi29 portal complexes (dots). The solid line represents the fit obtained by three component exponential fitting the exchange data (see materials and methods). Peptic fragments (A) 136–159, (B) 197–220, (C) 160–185, (D) 1–13, and (E) 288–308. Highest number on the y-axis represents the theoretical number of exchangeable amide protons.

Figure 3

Figure 3

Plots of the number of deuterium atoms incorporated according to the exchange period for peptic fragments from the Phi29 portal complexes (dots). The solid line represents the fit obtained by three component exponential fitting the exchange data (see materials and methods). Peptic fragments (A) 136–159, (B) 197–220, (C) 160–185, (D) 1–13, and (E) 288–308. Highest number on the y-axis represents the theoretical number of exchangeable amide protons.

Figure 4

Figure 4

Hydrogen/deuterium exchange profile map of the Phi29 portal complex. Hydrogen/deuterium exchange rates are categorized three groups, fast (red), intermediate (yellow), and slow (blue), based on the curve fitting results from a three component exponential model. Top (left panel) and side views (right panel) of the Phi29 portal complex crystal structure (PDB: 1IJG) are mapped according to exchange rates using PyMol. Regions where are not covered by hydrogen/deuterium exchange are colored grey.

Figure 5

Figure 5

Amino acid sequences of the P22 portal protein and hydrogen/deuterium exchange profile for peptide residues 632–658 and 309–335. (A) Amino acid sequence of the P22 portal protein. The observed peptic fragments of the P22 portal protein are underlined (blue lines). The peptic fragments, which were identified by a combination of exact mass matching and MS/MS sequencing, span ~90% of the protein primary sequence. The predicted elements of secondary structure were calculated and mapped onto the sequence as described in Figure 1. (B and C) Overlaid mass spectra of the peptic fragment corresponding to residues 632–658 and 309–335, respectively, from P22 portal protein monomer (black) and complex (red) at various exchange times. In each panel, the bottom spectrum represents the un-exchanged control.

Figure 5

Figure 5

Amino acid sequences of the P22 portal protein and hydrogen/deuterium exchange profile for peptide residues 632–658 and 309–335. (A) Amino acid sequence of the P22 portal protein. The observed peptic fragments of the P22 portal protein are underlined (blue lines). The peptic fragments, which were identified by a combination of exact mass matching and MS/MS sequencing, span ~90% of the protein primary sequence. The predicted elements of secondary structure were calculated and mapped onto the sequence as described in Figure 1. (B and C) Overlaid mass spectra of the peptic fragment corresponding to residues 632–658 and 309–335, respectively, from P22 portal protein monomer (black) and complex (red) at various exchange times. In each panel, the bottom spectrum represents the un-exchanged control.

Figure 5

Figure 5

Amino acid sequences of the P22 portal protein and hydrogen/deuterium exchange profile for peptide residues 632–658 and 309–335. (A) Amino acid sequence of the P22 portal protein. The observed peptic fragments of the P22 portal protein are underlined (blue lines). The peptic fragments, which were identified by a combination of exact mass matching and MS/MS sequencing, span ~90% of the protein primary sequence. The predicted elements of secondary structure were calculated and mapped onto the sequence as described in Figure 1. (B and C) Overlaid mass spectra of the peptic fragment corresponding to residues 632–658 and 309–335, respectively, from P22 portal protein monomer (black) and complex (red) at various exchange times. In each panel, the bottom spectrum represents the un-exchanged control.

Figure 6

Figure 6

Plots of the number of deuterium atoms incorporated according to the exchange period for peptic fragments from the P22 portal monomers (open triangles) and complexes (open circles). The error bars represent the standard deviation for three independent measurements. The solid line represents the fit obtained by three component exponential fitting the exchange data (see materials and methods). Peptic fragments (A) 164–186, (B) 632–658, (C) 246–262, (D) 309–335, (E) 57–78, and (F) 567–591. Highest number on the y-axis represents the theoretical number of exchangeable amide protons.

Figure 6

Figure 6

Plots of the number of deuterium atoms incorporated according to the exchange period for peptic fragments from the P22 portal monomers (open triangles) and complexes (open circles). The error bars represent the standard deviation for three independent measurements. The solid line represents the fit obtained by three component exponential fitting the exchange data (see materials and methods). Peptic fragments (A) 164–186, (B) 632–658, (C) 246–262, (D) 309–335, (E) 57–78, and (F) 567–591. Highest number on the y-axis represents the theoretical number of exchangeable amide protons.

Figure 6

Figure 6

Plots of the number of deuterium atoms incorporated according to the exchange period for peptic fragments from the P22 portal monomers (open triangles) and complexes (open circles). The error bars represent the standard deviation for three independent measurements. The solid line represents the fit obtained by three component exponential fitting the exchange data (see materials and methods). Peptic fragments (A) 164–186, (B) 632–658, (C) 246–262, (D) 309–335, (E) 57–78, and (F) 567–591. Highest number on the y-axis represents the theoretical number of exchangeable amide protons.

Figure 7

Figure 7

Overall pair-wise hydrogen/deuterium exchange comparison between the P22 portal monomers and complexes. Each bar represents the relative contributions of the three components fast (red), intermediate (yellow) and slow (blue) with exchange rate constants k1 (>1 min−1), k2 (0.01 min−1 – 1 min−1) and k3 (<0.01 min−1), respectively.

Figure 8

Figure 8

The crystal structure of the Phi29 connector was docked into the cryoEM reconstruction of the P22 portal as in Figure 1. Using the alignment of predicted helical regions of P22 with the known helical regions of Phi29 (as shown in Figure 1) the extent of exchange protection observed upon P22 portal assembly was mapped onto the structure. Blue represents peptides showing protection upon assembly, yellow represents no appreciable change upon assembly, and grey represents regions that lie outside of the putative conserved domain.

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