20 Research Frontiers 2019 Research Frontiers 2019 Life Science Currently, protein X-ray structures are usually determined from diffraction data of cryocooled crystals at around 100 K. It is believed that the structures at such cryogenic temperatures are basically identical to those at room temperatures where proteins actually function. However, thermodynamic analyses of conformational changes occurring in protein crystals have been very difficult by cryogenic X-ray crystallography, because the proteins lose their dynamics properties and temperature-dependent information is lost after cryocooling in exchange for protection from X-ray damage. Therefore, there has recently been much interest in the use of X-ray diffraction measurements at non-cryogenic temperatures to obtain dynamic structural information. Copper amine oxidases catalyze the oxidative deamination of various primary amines to produce the corresponding aldehydes (Fig. 1(a)) [1]. The active site contains a Cu 2+ ion and a protein-derived quinone cofactor, topaquinone (TPQ) (Fig. 1(b)). We have determined the crystal structures of a copper amine oxidase from Arthrobacter globiformis (AGAO) that had been reduced with substrate amines under anaerobic coenditions. The determined structures revealed that the reduced cofactor has two distinct states: a semiquinone radical form (TPQ sq ) and an aminoresorcinol form (TPQ amr ) having the copper- ligated “on-copper” and indirectly copper-contacting “off-copper” conformations, respectively [2]. Previous UV-vis spectral studies in solution revealed that the equilibrium between TPQ sq and TPQ amr is affected significantly by temperature and pH. The structural basis for the temperature- and pH-dependent equilibrium remains unresolved. To address this problem, we applied a temperature-controlled humid air and glue-coating (HAG) method to non- cryocooled crystals of AGAO at SPring-8 BL38B1 and thermodynamically analyzed the conformational change of TPQ in the crystal [3]. The HAG method was originally developed by JASRI [4] and has recently been improved to allow strict temperature control of protein crystals under anaerobic conditions [5]. To investigate temperature-dependent equilibrium changes of the AGAO crystal structure, we collected In crystallo thermodynamic analysis of catalytic reaction in bacterial copper amine oxidase Fig. 1. (a) Presumed catalytic mechanism of AGAO. TPQ ox , oxidative form of TPQ; TPQ ssb , substrate Schiff base of TPQ; TPQ psb , product Schiff base of TPQ; TPQ amr , aminoresorcinol form of TPQ; TPQ sq , semiquinone radical form of TPQ; TPQ imq , iminoquinone form of TPQ. (b) Overall structure of the AGAO dimer as a ribbon model. Enlarged view: stick model of the active site. The off-copper conformer (TPQ amr ) and on-copper conformer (TPQ sq ) are drawn in magenta and green, respectively. Fig. 2. Temperature dependence of TPQ sq /TPQ amr equilibrium. The TPQ conformation in the ethylamine- reduced AGAO crystal was examined at pH 6.0 by the temperature-controlled HAG method. (a) Assigned models of TPQ sq (green sticks) and TPQ amr (magenta sticks) are superimposed on the F o – F c omit maps (gray mesh) for residue 382 (TPQ) contoured at 3.5 σ , determined at the indicated temperatures. (b) Average occupancies of TPQ amr and TPQ sq at various temperatures are shown by magenta and green bars, respectively, with S.E. ( n ≥ 6), assuming that the sum of occupancies of TPQ amr and TPQ sq is 1.0. (c) van't Hoff plots for the TPQ sq / TPQ amr equilibrium in solution (black) and crystal (red). (a) (b) Reductive half-reaction Oxidative half-reaction H R H O OH Cu(II) Cu(II) Cu(II) Cu(II) Cu(II) Cu(I) TPQ psb TPQ amr TPQ sq (on-copper) TPQ ssb TPQ ox TPQ imq O 2 H 2 O 2 H 2 O H 2 O RCH 2 NH 3 + RCH 2 NH 3 + NH 3 NH 3 H 2 O RCHO N + OH – – – O O O O H R N O OH O 2 4 5 O H H N HO + + + H 2 N NH 2 OH Asp298 TPQ amr (off-copper) His433 Asn381 Met602 Val282 His592 His431 Tyr284 TPQ sq (on-copper) Tyr384 (a) (b) (c) 4°C 1.0 0.8 0.6 0.4 0.2 0.0 4 10 15 20 0.0034 0.0035 0.0036 – 2 – 1 0 1 2 10°C 20°C 15°C Temperature (°C) 1/ ( T /K) ln ( K eq ) TPQ amr /TPQ sq Contents 21 Research Frontiers 2019 Research Frontiers 2019 diffraction data of AGAO crystals anaerobically reduced with ethylamine at 4, 10, 15, and 20°C. Relative occupancies of each conformer provided K eq values (equilibrium constant for TPQ sq / TPQ amr ) that clearly indicate that the off-copper TPQ amr shifts to the on-copper TPQ sq as the temperature increases (Fig. 2(a,b)). Thermodynamic parameters obtained from a van’t Hoff plot of K eq values (Fig. 2(c)) for both crystalline ( Δ H º = 38 kJ/mol, Δ S º = 139 J/mol/K) and solution ( Δ H º = 26 kJ/mol, Δ S º = 83 J/mol/K) states indicated that the transition to TPQ sq is entropy-driven in both crystal and solution states. The in crystallo increases of Δ H ° and Δ S ° can be ascribed to the packing effect in the crystal lattice and suggest the reason for an equilibrium shift of TPQ sq / TPQ amr that is directly linkied to structural changes. To clarify pH-dependent equilibrium changes, the structures of 2-phenylethylamine (2-PEA)-reduced crystals were determined at 15°C with various pHs under aerobic conditions. The F o – F c omit maps for the TPQ moiety were considerably different depending on pH, indicating the formation of various intermediates (Fig. 3(a)). These differences suggest that the binding of the product aldehyde, phenylacetaldehyde (PAA), to the hydrophobic pocket facilitates the transition to TPQ sq at high pH and the formation of TPQ psb at low pH by the back reaction between TPQ amr and PAA in the crystals. (Fig. 3(b)). Our study revealed that the temperature-controlled HAG method has great advantages for detecting the equilibrium mixture of catalytic intermediates in protein crystals and will enable the elucidation of ‘ in crystallo ’ thermodynamics of conformational changes. We expect that the temperature-controlled HAG method will provide an important addition to various X-ray crystallographic techniques currently being developed. References [1] V.J. Klema et al .: Int. J. Mol. Sci. 13 (2012) 5375. [2] T. Murakawa et al .: J. Biol. Chem. 290 (2015) 23094. [3] T. Murakawa, S. Baba, Y. Kawano, H. Hayashi, T. Yano, T. Kumasaka, M. Yamamoto, K. Tanizawa, T. Okajima: Proc. Natl. Aca. Sci. USA 116 (2019) 135. [4] S. Baba et al .: Acta Crystallogr. D Biol. Crystallogr. 69 (2013) 1839. [5] S. Baba et al .: J. Appl. Cryst. 52 (2019) 699. Takeshi Murakawa a, *, Seiki Baba b and Toshihide Okajima c a Department of Biochemistry, Osaka Medical College b Japan Synchrotron Radiation Research Institute (JASRI) c Institute of Scientific and Industrial Research, Osaka University *Email: murakawa@osaka-med.ac.jp Fig. 3. pH dependence of equilibrium. (a) pH profile of 2-PEA-reduced AGAO crystal structures at 15°C at various pH determined by the temperature-controlled HAG method. The determined active-site structures are superimposed on the F o – F c omit map (gray mesh) for residue 382 and the product aldehyde contoured at 3.5 σ . Residue 382 is represented by a stick model with the same color scheme as in the bar graph. Average occupancies of the intermediates are shown by bars with S.E. ( n ≥ 4): cyan, cis -TPQ psb ; brown, trans -TPQ psb ; purple, TPQ amr ; green, TPQ sq . (b) Possible mechanism of pH-dependent equilibrium changes in the 2-PEA-reduced AGAO crystal. (a) (b) 6.0 D298 D298 D298 D298 + 2-PEA D298 D298 O OH OH OH OH H H H H N + N + O – – H 2 O H 2 N ¨ H 2 N ¨ – H 2 O H 2 N NH 2 + H 2 O + H + + H + Cu 2+ Cu + + O O O O O O O O O OH OH OH HO HO HO HO H H H H H Si face Re face O O O – O – O – O – O – 0.0 0.2 0.4 0.6 0.8 1.0 7.0 8.0 9.0 10.0 pH Occupancy pH ≤ 7 cis -TPQ psb TPQ amr TPQsq TPQ ox TPQ ssb TPQ psb trans -TPQ psb pH ~ 7 pH > 8
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