ATP-binding cassette (ABC) multidrug transporters are membrane proteins which transport various structurally unrelated substrates using the energy of ATP hydrolysis. The precise transport mechanisms of a human ABC multidrug transporter, P-glycoprotein (hP-gp), are not fully understood based on the crystal structure, because of the difficulty of crystallization of hP-gp. Recently, we have determined the crystal structures of a hP-gp homolog, CmABCB1 from Cyanidioschyzon merolae, at 2.6 Å, and its complex with a novel allosteric inhibitor at 2.4 Å. Here, we present how these high resolution structure determinations were achieved, and explain the detailed architecture of the transmembrane domains of CmABCB1.
Structural determination of membrane proteins is a key step to understand the molecular mechanisms of the membrane proteins. Lipidic cubic phase (LCP) crystallization is a technique to crystallize the membrane proteins in lipid environment and one of the effective approaches for the membrane protein crystallization. In this article, we describe the overview of crystallization of the membrane proteins in LCP, including the basic principles and procedures of LCP crystallization.
Crystallographic investigation combined with QM/MM simulation and biochemical analysis is the powerful tool to elucidate the enzyme mechanisms. Here, orotidine 5'-monophosphate decarboxylase (ODCase) is selected as a representative example of intensively investigated enzymes. This enzyme accelerates the conversion of orotidine 5'-monophosphate into uridine 5'-monophosphate by 17 orders of magnitude, and is known as one of the most proficient enzymes. Although the decarboxylation reaction is an electrophilic-like reaction, ODCase also catalyzes nucleophilic-like reactions at the same catalytic site with the decarboxylation. Our crystallographic analysis at 1.0−1.8 Å resolution combined with ab initio calculation and biochemical assays elucidated the catalysis of ODCase utilizes the substrate distortion in addition to the transition state stabilization.
X-ray free-electron laser (XFEL) source produces X-ray pulses more than a billion times brighter than the most powerful synchrotron source. The extremely intense X-ray pulses emerging from XFEL source, along with the appearance of serial femtosecond X-ray crystallography (SFX) technique enabled us to reveal radiation damage-free protein structures from nano-/micro-crystals. Current state of protein structure analyses using XFEL is described.
Small angle X-ray scattering (SAXS) analysis is a facile and useful method to determine structures of biological macromolecules in solution. Although SAXS analysis generally provides significant information of molecular shape at low resolution, in combination with other biophysical techniques such as X-ray crystallography, SAXS analysis exhibits tremendous power for structural studies of biological macromolecules. In this review, we described our recent studies of SAXS analysis for biological macromolecules (BioSAXS) and future perspective for integrated structural biology using BioSAXS.
Today, we can determine the structural basis of a biological molecule by X-ray crystallography or X-ray solution scattering. However, static structures do not say a lot for the realtime transition of the functional dynamics. Here we introduce the pump-probe method using combined single bunch X-ray from synchrotron source and pulsed laser system in sub nanosecond time resolution. Time-resolved X-ray solution scattering measurement of dimeric hemoglobin revealed a spiral motion of two subunits and three intermediate states. The pump-probe method is even powerful technique in the other methods of measurement for the protein dynamics.
X-ray diffraction technique is suitable for in-situ observation and quantitative analysis of phenomena occurring in manufacturing process of industries. Its time resolution has been progressed by applying synchrotron radiation. Two cases of industrial application of synchrotron radiation are introduced.
We report our recent approach to the direct structure analysis of X-ray crystal truncation rod scattering data for thinfilm interface structures. Our approach is a combination use of a holographic method and the iterative phase-retrieval methods, which are respectively used for the direct construction of the initial structure model and the final refinement of the model. Results of the direct structure analysis for a Bi thinfilm and Bi/Bi2Te3 topological insulator thinfilm are presented. The holographic method successfully imaged out interfacial wetting layers for both the systems. Structural parameters were derived quantitatively by using the phaseretrieval methods, which are relevant to understanding electronic properties and film growth mechanisms at the interfaces.
Heme oxygenase（HO）is a unique enzyme that catalyzes the conversion of heme to biliverdin,carbon monoxide and free iron. The enzyme is present in not only mammal but also plant, algae and pathogenic bacteria. In order to understand mechanisms of the substrate binding and the product release of bacterial HO, we have determined the crystal structures of the substratefree, Fe3+-biliverdin-bound, biliverdin-bound forms and reaction intermediates between the latter two states of HmuO, a heme oxygenase from Corynebacterium diphtheriae. In addition to these high resolution structures, we have conducted molecular dynamics simulation for the hemebinding and bilivedin-release. The substrate-free HmuO shows a widely open active site which is formed by a partially unwounded α-helix. The water molecule cluster is rearranged when the substrate is bound to HmuO. Upon reduction of Fe3+ to Fe2+, the axial histidine dissociates from Fe2+, followed by the relase of Fe2+ from the biliverdin group. The water molecule comes into the resulted space and forms hydrogen bonds between the axial histidine and the substrate biliverdin. From these results, we can discuss the molecular mechanism of HmuO at atomic level.