A docking model based on mass spectrometric and biochemical data describes phage packaging motor incorporation

Abstract

The molecular mechanism of scaffolding protein-mediated incorporation of one and only one DNA packaging motor/connector dodecamer at a unique vertex during lambdoid phage assembly has remained elusive because of the lack of structural information on how the connector and scaffolding proteins interact. We assembled and characterized a phi29 connector-scaffolding complex, which can be incorporated into procapsids during in vitro assembly. Native mass spectrometry revealed that the connector binds at most 12 scaffolding molecules, likely organized as six dimers. A data-driven docking model, using input from chemical cross-linking and mutagenesis data, suggested an interaction between the scaffolding protein and the exterior of the wide domain of the connector dodecamer. The connector binding region of the scaffolding protein lies upstream of the capsid binding region located at the C terminus. This arrangement allows the C terminus of scaffolding protein within the complex to both recruit capsid subunits and mediate the incorporation of the single connector vertex.

Fig. 1.

The x-ray crystal structures of connector protein (Protein Data Bank code 1FOU, chains A and B) (A) and scaffolding protein (Protein Data Bank code 1NO4, chains A and B) (B).

Fig. 2.

Native mass spectra of connector-scaffolding complexes. The mass spectra of connector-scaffolding complexes formed at 25- (A), 10- (B), and 2-fold (C) molar excess of scaffolding protein are shown. The number of scaffolding protein molecules present in the assigned connector-scaffolding complexes is indicated above the peaks in the mass spectra. Bar graphs of the relative abundance of the connector-scaffolding species at 2-, 10-, and 25-fold molar excess of scaffolding protein (white, light gray, and dark gray, respectively) determined experimentally (D) and calculated assuming a K_D of 25 μm (E) are shown. Complexes with more than 12 scaffolding proteins bound to a connector dodecamer were not detected. Peaks around m/z = 12,000 and 13,000 in C correspond to dimers of the connector dodecamer.

Fig. 3.

Identification of connector binding region of scaffolding protein. A, the ability of scaffolding deletion mutants to solubilize connector protein at low ionic strength buffer was assayed by SDS-PAGE following centrifugation. The amounts of input and insoluble protein are shown in the odd and even lanes, respectively. Lanes 1 and 2, connector protein only; lanes 3 and 4, connector plus wild type (WT) scaffolding protein; lanes 5 and 6, connector plus Δ74–98 scaffolding protein; lanes 7 and 8, connector plus Δ70–98 scaffolding protein. B, the effect of temperature on the ability of wild type and temperature-sensitive mutant scaffolding protein to solubilize connector protein. The fraction of connector solubilized following incubation at 20, 30, and 42 °C was quantified by centrifugation and SDS-PAGE for connector protein alone (closed squares) or connector in the presence of wild type (open squares) or S65N scaffolding protein (open triangles).

Fig. 4.

Identification of cross-linked peptides derived from connector-scaffolding complex. A, SDS-PAGE separation of cross-linked connector protein (lane 1) and connector-scaffolding complexes (lane 2). Connector monomer (Conn/M), connector dimer (Conn/D), and the connector-scaffolding species (CS1 and CS2) resulting from cross-linking are indicated with arrows. Molecular weight markers are in lane M. B, the MS/MS spectrum of the cross-linked peptide m/z = 660.7504 (5+ charge). The assigned b-ions and y-ions generated by fragmentation of the m/z = 660.8 parent ion and their corresponding masses are labeled. The inset shows the cross-linked fragments with the identified b- and y-ions labeled. C, the MS/MS spectrum of the cross-linked peptide m/z = 731.3822 (3+ charge). The assigned b-ions and y-ions generated by fragmentation of the m/z = 731.4 parent ion and their corresponding masses are labeled. The inset shows the cross-linked fragments with the identified b- and y-ions labeled.

Fig. 5.

Identification of cross-link peptide in mutant complex. The complex formed between mutant scaffolding protein D58K and wild type connector was cross-linked. A unique band in the SDS-PAGE was in-gel digested and yielded a unique parent ion of m/z = 876.7647 (6+ charge) that was subjected to MS/MS. The series of b- and y-ions generated by fragmentation and their assignments are indicated and mapped onto the sequence of the cross-linked peptide.

Fig. 6.

Docking model of connector-scaffolding complexes. The docking model showing six scaffolding dimers (blue) docked to the wide domain interface between two adjacent connector subunits (green) in the dodecamer. The N-terminal arrowhead motif of the scaffolding points upward.

Fig. 7.

Model of connector-scaffolding complex docked in procapsid. The docking model of the connector-scaffolding complex was fit into the connector vertex of a cryo-EM reconstruction of the procapsid (blue). The crystal structures of scaffolding (orange), connector (yellow), fitted HK97 capsid proteins (red, green, and blue), and packaging RNA (pink) are shown as ribbon diagrams. Interacting residues from the connector and the scaffolding are shown as van der Waals spheres colored by element (gray for carbon, blue for nitrogen, and red for oxygen). This figure was generated using CHIMERA (42).