# NMR Solution Structure of the N‐Terminal GSPII Domain from the Thermus Thermophilus Traffic ATPase PilF and Reconstruction of its c‐di‐GMP Binding Capability

## TL;DR

This study reveals why a specific GSPII domain from Thermus thermophilus PilF cannot bind c-di-GMP and how mutations can restore this ability.

## Contribution

The study identifies structural and sequence-based reasons for c-di-GMP binding failure in GSPII-A and demonstrates how to restore it through mutagenesis.

## Key findings

- GSPII-A lacks c-di-GMP binding due to sequence deviations, a stabilizing helical capping motif, and intersubdomain interactions.
- Mutagenesis and truncations can restore c-di-GMP binding in GSPII-A.
- Sequence variations in GSPII domains are crucial for functional diversification.

## Abstract

The cyclic dinucleotide c‐di‐GMP is an important second messenger molecule in bacteria and interacts with a variety of receptor molecules including RNA and protein domains. An important class of c‐di‐GMP‐binding protein domains are the general secretory pathway type II (GSPII) domains as exemplified by the N‐terminal domain of the ATPase MshE from Vibrio cholerae (MshEN). MshEN binds monomeric c‐di‐GMP via two consecutive copies of a 24‐residue sequence motif, which form a compact 4‐α‐helical bundle. The ATPase PilF from Thermus thermophilus regulates pilus formation, motility and DNA‐uptake. Its N‐terminal section contains three consecutive GSPII domains (GSPII‐A‐GSPII‐C) all with considerable sequence homology to MshEN. While the GSPII‐B and the GSPII‐C domains bind c‐di‐GMP, the GSPII‐A domain does not. To determine why it is incapable of c‐di‐GMP‐binding we determined the NMR‐solution structure of this domain. Our structure shows how small deviations in the consensus motif sequence, a stabilizing N‐terminal helical capping motif and intersubdomain interactions absent in MshEN cooperate to prevent c‐di‐GMP‐binding. By combining point mutations and truncations, we re‐established the c‐di‐GMP binding capability. Our findings shed new light on the evolution and functional diversification of GSPII domains and the importance of sequence variations for protein activity in this domain family.

GSPII‐A is the first of three consecutive GSPII domains of PilF from Thermus thermophilus and the only one that does not bind c‐di‐GMP. In this study we solve the structure of GSPII‐A and show the key factors hindering c‐di‐GMP binding. Through mutagenesis, we elucidate the minimal motif conservation needed for c‐di‐GMP recognition.