Biochem. J. (2012) 448, 201–211 (Printed in Great Britain) doi:10.1042/BJ20120668 201 Structural insight into how Pseudomonas aeruginosa peptidoglycan- hydrolase Tse1 and its immunity protein Tsi1 function Guijun SHANG* 1 , Xiuhua LIU*† 1 , Defen LU* 1 , Junbing ZHANG*, Ning LI*, Chunyuan ZHU*‡, Shiheng LIU*, Qian YU*, Yanyu ZHAO*, Heqiao ZHANG§, Junqiang HU*, Huaixing CANG§, Sujuan XU* and Lichuan GU* 2 *State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China, †College of Life Sciences, Hebei University, Baoding 071002, China, ‡State Key Laboratory of Crop Biology, Shandong Agricultural University, Taian 271018, China, and §Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China Tse1 (Tse is type VI secretion exported), an effector protein pro- duced by Pseudomonas aeruginosa, is an amidase that hydrolyses the γ -D-glutamyl-DAP (γ -D-glutamyl-L-meso-diaminopimelic acid) linkage of the peptide bridge of peptidoglycan. P. aeruginosa injects Tse1 into the periplasm of recipient cells, degrading their peptidoglycan, thereby helping itself to compete with other bacteria. Meanwhile, to protect itself from injury by Tse1, P. aeruginosa expresses the cognate immunity protein Tsi1 (Tsi is type VI secretion immunity) in its own periplasm to inactivate Tse1. In the present paper, we report the crystal structures of Tse1 and the Tse1-(6–148)–Tsi1-(20-end) complex at 1.4 Å and 1.6 Å (1 Å = 0.1 nm) resolutions respectively. The Tse1 structure adopts a classical papain-like α + β fold. A cysteine–histidine catalytic diad is identified in the reaction centre of Tse1 by structural comparison and mutagenesis studies. Tsi1 binds Tse1 tightly. The HI loop (middle finger tip) from Tsi1 inserts into the large pocket of the Y-shaped groove on the surface of Tse1, and CD, EF, JK and LM loops (thumb, index finger, ring finger and little finger tips) interact with Tse1, thus blocking the binding of enzyme to peptidoglycan. The catalytic and inhibition mechanisms provide new insights into how P. aeruginosa competes with others and protects itself. Key words: amidase, interbacterial competition, type VI secretion exported 1 (Tse1), type VI secretion immunity 1 (Tsi1), type VI secretion system (T6SS). INTRODUCTION To manipulate their invasion into host cells or the environment, pathogenic bacteria have developed several secretion systems which transport corresponding effector proteins to the exterior of bacteria. At least six types of secretion machines, termed T1SS–T6SS (type I to type VI secretion systems) respectively, have been identified in the Gram-negative bacterial pathogens of animals and plants [1–3]. These secretion machines have versatile structural components and substrates, and are crucial for the interactions between host and bacteria, as well as different types of bacteria [4,5]. T1SS, T2SS and T4SS usually transport effectors into the bacterial cell surface or into the milieu [6,7]. The T3SS and T4SS secretion systems of many bacterial pathogens take part in delivering secretion proteins into eukaryotic cells, yielding spectacular cellular responses, such as reshuffling of host cell actin and hijacking of host signalling transduction, and eventually they subvert the host response [8–12]. The characterized T6SS has emerged as a novel molecular syringe in many bacterial species [13–15] and plays roles in virulence-related processes, interbacterial competition and biofilm formation [1,16–20]. Although T6SSs are involved in numerous processes of bacterial life cycles, their underlying mechanisms are still unknown. Additionally, the secreted effector proteins, which directly interact with host cell or competitive bacteria, have become increasingly attractive for the attention of biologists because they can cause so many physiological consequences to cells. Three effectors have been identified in the opportunistic pathogen Pseudomonas aeruginosa, namely Tse1, Tse2 and Tse3 (Tse is type VI secretion exported). These three effectors are controlled by the H1-T6SS of P. aeruginosa and injected into the cytoplasm (Tse2) and periplasm (Tse1 and Tse3) of the recipient. Tse2 is a toxic effector. Its main physiological function is correlated with growth arrest in prokaryotic cells, providing a competitive advantage for P. aeruginosa during fierce niche competition. To avoid inhibiting itself, an immunity protein Tsi2 (Tsi is type VI secretion immunity) in the cytoplasm of P. aeruginosa acts as a Tse2 inhibitor. Bacteria lacking Tsi2 are vulnerable and are easily excluded during intrabacterial competition [17]. Tse1 has amidase activity hydrolysing the peptides between γ -D-glutamyl-DAP (γ -D-glutamyl-L-meso- diaminopimelic acid) of the murein, and Tse3 has muramidase activity cutting the chain of glycan of the murein. They are both lytic enzymes that degrade peptidoglycan. To protect itself from accidental injury via contact with other P. aeruginosa cells, the bacterium secretes two T6SS immunity proteins (Tsi1 and Tsi3) into its own periplasm, binding and neutralizing the cognate toxin. These three effectors and their cognate immunity proteins exist exclusively in P. aeruginosa. Therefore they inhibit the growth of competitor cells without causing accidental injury to P.aeruginosa, providing fitness advantages in the microbial jungle competition [20,21]. In the present paper, we report the crystal structures of Tse1 and the Tse1-(6–148)–Tsi1-(20-end) complex. The structural data, combined with biochemical data, provide further insight into the mechanism by which Tse1 functions as an amidase and how it is controlled by its cognate protein Tsi1. Abbreviations used: DLS, dynamic light scattering; IPTG, isopropyl β-D-thiogalactopyranoside; LB, Luria–Bertani; PEG, poly(ethylene glycol); RMSD, root mean square deviation; SeMet, selenomethionine; SLP, surface layer protein; SPR, surface plasmon resonance; TEV, tobacco etch virus; Tse, type VI secretion exported; Tsi, type VI secretion immunity; T1SS etc., type I secretion system etc. 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed (email lcgu@sdu.edu.cn). The atomic co-ordinates and structure factors have been deposited in the PDB with the accession codes 4EQ8 for Tse1 and 4EQA for the Tse1-(6– 148)–Tsi1-(20-end) complex. c  The Authors Journal compilation c  2012 Biochemical Society Biochemical Journal www.biochemj.org