Photon Factory Activity Report 2016 #34 (2017)

AR-NW12A/2016G657

X-ray Crystallographic Analysis of M16F/T36K Double Mutant of Pseudoazurin
Takahide Yamaguchi,1,2 Naoki Takebayashi,1 Kohei Akao,1 Chihiro Sakai1 and Takamitsu
Kohzuma1,2*1Inst. Quantum Beam Sci., Ibaraki University, 2-1-1 Bunkyo, Mito, 310-8512, Japan
2Frontier Appl. Atomic Sci. Res. Centre, Ibaraki University, 162-1 Shirakata, Tokai, 319-1106, Japan
1 Introduction
The structure and function of active sites in protein isdetermined by not only the immediate molecularenvironment but also the outer sphere interactions, suchas electric dipole, hydrogen bond and so on. In
metalloproteins, the regulations of Cu site properties
through the modifications of hydrogen bonds orhydrophobicity of coordination environment werereported by Lu and his co-workers [1]. We also reported
the importance of hydrogen bond network for theelectronic structure of heme Fe in cytochrome c’ [2].
Non-covalent weak interaction due to the dispersion force
between amino acid residues are also known to attends
the structure-and-function relationship of proteins,
however, the rational design of active site properties
depends on the outer sphere environment involving theweak interactions, which have never been reported to date.
Pseudoazurin (PAz) has the redox-active Cu site for abiological electron transfer. The second-sphere noncovalent weak interaction in the PAz has been studied bystructure analysis, several spectroscopic methods,electrochemistry, and theoretical approach [3]. The highresolution X-ray crystal structure analysis of M16F/T36K
double mutant PAz was performed at Photon Factory toknow of the additivity of spectroscopic/electrochemicalproperties for each single mutation, M16F and T36K.

coordinated H81. The structure in the double mutant,
M16F/T36K is consistent with M16F single mutant [5].
Interestingly the mutation of T36 to lysine residue gavethe structural rearrangement of hydrogen-bonding
networks around H6 (Figure 1). The hydrogen bond
between H6 and T36 recognized in the structure of WT
protein is disappeared in the double mutant protein. A
new hydrogen bond between H6 and E4 is observed
instead of the H6-T36 hydrogen bond in the M16F/T36K.
The other rearrangements of hydrogen bonds are also
induced by the movement of P35, I34, and F33 of thedouble mutant PAz. The relevant studies for the additivityof spectroscopic/electrochemical properties for the double
mutant are currently in progress based on the X-ray
crystal structure analysis of M16F/T36K double mutant.

Active Site

Thr36(WT)
Lys (M16F/T36K)
Pro35

2.66 Å: H-bond(WT)

His6
Ile34

2 Experiment
The protein of M16F/T36K variant was expressed in E.
coli and purified. The crystal of M16F/T36K variant wasobtained by the hanging drop vapour diffusion method.
The single crystal X-ray diffraction data for theM16F/T36K variant were collected at beam line NW 12A(PF-AR). The temperature of crystal was 100 K. The
space group of M16F/T36K variant was C2 space group,and the cell parameters were a = 93.2 Å, b = 41.2 Å, c =34.8 Å, a = g = 90.0∘, b = 98.7∘. The structure ofM16F/T36K variant was refined at 1.19 Å resolution.
(PDB ID: 5XMO)
3 Results and Discussion
The structure of M16F/T36K PAz at 1.19 Å resolution
demonstrated the co-existence of two Cu position, which
two conformers were assigned to the axial and rhombic
structure in the Wild Type (WT) PAz as we reported
previously [4]. Several structural changes are recognizedat the mutated amino acid residues in the outer sphere
region of M16 and T36 residues. The replacement of M16
to phenylalanine provides π-π interaction with copper

Phe33

2.82 Å: H-bond(M16F/T36K)

Glu4

Fig. 1: The rearrangement of hydrogen bond around His6
and flipping of Pro35 between wild type (green) andMet16Phe/Thr36Lys (blue).
References[1] N. M. Marshall et al., Nature 462, 113 (2009).
[2] A. Takashina et al., Bull. Chem. Soc. Jpn., 90, 169(2017).
[3] R. F. Abdelhamid et al., J. Biol. Inorg. Chem. 12, 165(2007).
[4] T. Yamaguchi et al., RSC Adv., 6, 88358 (2016)[5] S. Yanagisawa et al., J. Am. Chem. Soc., 130, 15420(2008).
* takamitsu.kohzuma.qbs@vc.ibaraki.ac.jp