Photon Factory Activity Report 2014 #32 (2015) B

BL-17A, 5A, NE3A, NW12A/2013G026

Crystal structure analysis of peptide ligase PGM1 from Streptomyces cirratus.
Takashi Matsui1, Motoyoshi Noike2, Shouta Ohtaki1, Tohru Dairi2 and Hiroyuki Morita1,*1
Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan2
Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan
1 Introduction
Peptide antibiotics form a large subclass of naturalproducts, including clinically important pharmaceuticals.
Streptomyces cirratus produces structurally uniquepheganomycins (PGMs), which show anti-mycobacteriaactivity. PGM contains a nonproteinogenic amino acidand a proteinogenic core peptide. The biosynthetic genecluster from S. cirratus responsible for PGM production
was identified [1]. PGM1 was found in the cluster as
potentially capable of linking between the precursor
peptides and the nonproteinogenic amino acid withguanidinoacetic acid scaffold. PGM1 also accepted avariety of peptides as the nucleophile. To clarify theligation mechanism between amino acid and peptide andunderstand the substrate’s promiscuity, PGM1 structure
was determined.
2 Experiment
Crystallization – 20 mg/ml selenomethionine-labeled
PGM1 with or without adenylyl imidodiphosphate(AMPPNP) were crystallized with sitting-drop vapor
diffusion method. 2 µl of PGM1 and 2 µl of reservoir
solution were mixed, and equilibrated against 500 µl ofreservoir solution at 20ºC. Diffraction-quality apo crystals
were finally obtained in 0.1 M Tris-HCl, pH 8.3, 1.2 M
lithium sulfate, and 10 mM L-methionine after a few days
incubation. PGM1 with AMPPNP was crystallized 0.1 M
Tris-HCl, pH 8.1, 1.2 M lithium sulfate and 10 mM
AMPPNP after a few days incubation.
Data collection and structure determination – Crystals
were transferred into the reservoir solution containing
10%(v/v) glycerol as a cryoprotectant, and were flashfrozen in a nitrogen stream. Single-wavelength
anomalous diffraction (SAD) data of PGM1 apo andAMPPNP complex crystals were collected at beamline
BL-17A and BL-5A under cryogenic condition at -173ºC,
respectively. Wavelength of 0.97939 Å at BL-17A and0.97901 Å at BL-5A were used for data collection on thebasis of the fluorescence spectrum of the Se K absorption
edge. The diffraction data was processed and scaled usingXDS. Se sites were determined and refined and the initial
phase of PGM1 was calculated with AutoSol in PHENIX.
The initial phase of PGM1-AMPPNP complex wascalculated by the molecular replacement (MR)-SAD
method with AutoSol in PHENIX using PGM1 apo as thesearch model. The structure was rebuilt using AutoBuildin PHENIX, modified manually with Coot and refinedwith phenix.refine in PHENIX. The coordinates andstructure factors have been deposited under accession

number 3WVQ for the PGM1 apo, and 3WVR for thePGM1-AMPPNP complex.
3 Results and Discussion
X-ray structures of PGM1 apo and AMPPNP complex
were refined at 1.96 and 2.17 Å resolutions, respectively.
The final R-values of apo and complex structures were
19.4% and 20.2% (Rfree = 23.1% and 24.6%), respectively.
Asymmetric unit contains four monomers, which form
the two set of biologically active symmetric dimers.
PGM1 consisted of four domains to form a central core
(N, A, and C domains) and a upper (B domain) structures
as seen in those of the ATP-grasp superfamily enzymes(Fig 1a). The central core and upper structures formed alarge cavity at the center of the PGM1 structure. TheATP-binding site was located deeply inside the cavity.
Most of the conserved resides lining PGM1’s ATPbinding site were in a location and orientation similar tothat in other ATP-grasp enzymes. PGM1 also formed along cleft from the large cavity. The large cavity and thecleft created an unusual active site (Fig 1b). The sitedirected mutagenesis stidies of E255, S316, R335, andS339 formed the cavity and those of A14 and S190
formed the cleft were suggested that the large cavity andthe long cleft are enable to accept the guanidinoacetic
acid scaffold substrates and the several peptides.

Fig. 1: Crystal structure of S. cirratus PGM1. (a) Overall
structure of PGM1-AMPPNP complex. The electron
densities of β- and γ-phosphates of AMPPNP were weak.
Thus, the structure was built as PGM1-AMP complex. (b)Close-up view of the active site. The bottom of the large
cavity and the cleft are highlighted in blue and orange,
respectively.
References[1] M. Noike et al., Nat. Chem Biol. 11, 71-76 (2015).
* hmorita@inm.u-toyama.ac.jp