Abstract

P4 is a mobile genetic element (MGE) that can exist as a plasmid or integrated into its Escherichia coli host genome, but becomes packaged into phage particles by a helper bacteriophage, such as P2. P4 is the original example of what we have termed molecular piracy, the process by which one MGE usurps the life cycle of another for its own propagation. The P2 helper provides most of the structural gene products for assembly of the P4 virion. However, when P4 is mobilized by P2, the resulting capsids are smaller than those normally formed by P2 alone. The P4-encoded protein responsible for this size change is called Sid, which forms an external scaffolding cage around the P4 procapsids. We have determined the high-resolution structure of P4 procapsids, allowing us to build an atomic model for Sid as well as the gpN capsid protein. Sixty copies of Sid form an intertwined dodecahedral cage around the T = 4 procapsid, making contact with only one out of the four symmetrically non-equivalent copies of gpN. Our structure provides a basis for understanding the sir mutants in gpN that prevent small capsid formation, as well as the nms super-sid mutations that counteract the effect of the sir mutations, and suggests a model for capsid size redirection by Sid.

Highlights

  • We produced P4 procapsids in vitro from co-expressed gpN, protease-deficient mutant gpO, and size determination protein (Sid)

  • Atomic models of gpN built into a cryo-electron microscopy single particle reconstruction of the P4 procapsid later revealed that the sir loop of gpN interacts with Sid, suggesting that the sir and nms mutations disrupt and restore, respectively, Sid binding to gpN in the procapsid [4]

  • In honor of Dr Michael Rossman and his research on the unusual phage φX174, which assembles its procapsid with an external scaffolding protein, we present here our structure of the Sid external scaffolding protein and its mechanism of capsid size redirection

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Summary

Introduction

We produced P4 procapsids in vitro from co-expressed gpN, protease-deficient mutant gpO, and Sid. Previous studies identified size responsiveness (sir) mutations clustered in a region of gpN denoted the sir loop that allow P2 to escape size redirection. N mutation sensitive (nms), or “super-Sid”, mutations localized near the C-terminus of Sid act as second-site suppressors of the sir mutations and restore size redirection [1,4].

Results
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