Structural and functional insights into the distinct DNA recognition mechanisms of the terminase small subunit TerS from cyanophages

Abstract

Efficient genome packaging is a critical step in the phage life cycle, directly influencing viral maturation and infectivity. In tailed phages, this process is driven by a packaging motor composed of a portal protein and a terminase complex. The terminase complex usually consists of a large subunit (TerL) and a small subunit (TerS), which cooperate to recognize, cleave, and translocate genomic DNA into the capsid. However, due to the remarkable diversity and complexity of phage packaging systems, the molecular mechanisms governing TerS-mediated DNA recognition remain poorly understood. Here, we report the 3.51 Å cryo-electron microscopy structure of the TerS from the short-tailed cyanophage Pam5, which infects the host Pseudanabaena mucicola Chao 1806. Pam5 TerS assembles into a nonameric ring with a radially symmetric spiral architecture. Biochemical assays show that Pam5 TerS recognizes the genomic DNA via a specific interaction between the N-terminal helix-turn-helix (HTH) domain of TerS and a 21-bp DNA sequence within the terS gene. In contrast, the TerS from another short-tailed cyanophage, Pam1, which infects the same host, binds to DNA in a sequence-independent manner. These findings reveal that cyanophages, even infecting the same host, could adopt two distinct DNA recognition strategies: HTH-mediated sequence-dependent or sequence-independent modes. This work provides structural and mechanistic insights into the diverse DNA-recognition strategies of TerS and advances our understanding of the evolutionary plasticity of viral genome packaging mechanisms.

1. Introduction

Efficient genome packaging is a pivotal event in the bacteriophage life cycle, directly determining the viral maturation and infectivity [1]. In tailed bacteriophages, genome packaging is powered by a highly efficient molecular motor composed of a portal protein and a terminase complex capable of generating forces exceeding 50 pN [2]. The terminase complex typically comprises two components, the large subunit (TerL) and the small subunit (TerS). They usually assemble into ring-like oligomers that interact with each other, although their precise stoichiometry within the complex remains unclear [3].

The phage capsid contains a specialized vertex occupied by a dodecameric portal that plays essential roles in procapsid assembly, genome packaging, and viral maturation [4]. During DNA packaging, the terminase complex docks onto the portal, enabling the translocation of genomic DNA through the central channel of the portal into the capsid [5,6]. Once genome packaging is completed, the terminase complex dissociates from the portal, and the tail apparatus attaches to the portal vertex, thereby generating the mature virions [3]. TerL functions as the catalytic core of the packaging machinery, which cleaves the genomic DNA and drives its translocation into the capsid [7]. TerL comprises an N-terminal ATPase domain and a C-terminal nuclease domain, which are linked by a flexible linker [8]. In contrast, TerS was proposed to recognize the genomic DNA that ensures specific initiation of genome packaging [9]. The TerS proteins typically assemble into octameric to dodecameric rings [10,11], which transiently associate with TerL and modulate its ATPase and nuclease activities [11–13]. Despite a similar oligomeric architecture, TerS proteins display substantial sequence diversity, and in many phages, it remains unclear whether a terS gene is present at all [14,15].