Biophysics Reports

2016 Vol. 2, No. 2-4

Cover Story

Virus 3D atomic structures provide insight into our understanding of viral life cycles and the development of antiviral drugs. X-ray crystallography and cryo-EM have been used to determine the atomic structure of viruses. However, limited availability of biological samples, biosafety issues due to virus infection, and sometimes inherent characteristics of viruses, pose difficulties on combining both methods in determining viral structures. These have made solving the high resolution structure of some medically important viruses very challenging. The authors describe their recently employed protocols for determining the high-resolution structure of the virus-like particle of hepatitis E virus (HEV), a pathogen of viral hepatitis in human. These protocols include utilizing recombinant baculovirus system to generate sufficient amount of virus particles, single-particle cryo-EM to get an intermediate resolution structure as a phasing model, and X-ray crystal-lography for final atomic structure determination. The protocols have solved the hepatitis E virus structure to the resolution of 3.5Å. The combined methodology is generally applicable to other human infectious viruses.

Select articles

Previous Issue | Next Issue

Uniporter substrate binding and transport: reformulating mechanistic questions

Xuejun C. Zhang, Lei Han

2016, 2(2-4): 45-54. doi: 10.1007/s41048-016-0030-7

Abstract(68)

PDF(137)

Abstract:
Transporters are involved in material transport, signaling, and energy input in all living cells. One of the fundamentalquestionsabout transportersisconcernedwiththepreciseroleoftheirsubstrateindrivingthe transport process. This is particularly important for uniporters, which must utilize the chemical potential of substrateastheonlyenergysourcedrivingthetransport.Thus,uniporterspresentanexcellentmodelforthe understanding of how the difference in substrate concentration across the membrane is used as a driving force. Local conformational changes induced by substrate binding are widely considered as the main mechanism to drive the functional cycle of a transporter; in addition, reducing the energy barrier of the transition state has also been proposed to drive the transporter. However, both points of view require modification to allow consolidation with fundamental thermodynamic principles. Here, we discuss the relationship between thermodynamics and kinetics of uniporters. Substrate binding-induced reduction of the transition-state energy barrier accelerates the transport process in kinetic terms, while the chemical potential of the substrate drives the process thermodynamically.

Structure determination of a human virus by the combination of cryo-EM and X-ray crystallography

Zheng Liu, Tom S. Y. Guu, Jianhao Cao, Yinyin Li, Lingpeng Cheng, Yizhi Jane Tao, Jingqiang Zhang

2016, 2(2-4): 55-68. doi: 10.1007/s41048-016-0027-2

Abstract(128)

PDF(161)

Abstract:
Virus 3D atomic structures provide insight into our understanding of viral life cycles and the development of antiviral drugs. X-ray crystallography and cryo-EM have been used to determine the atomic structure of viruses. However, limited availability of biological samples, biosafety issues due to virus infection, and sometimes inherent characteristics of viruses, pose difficulties on combining both methods in determining viral structures. These have made solving the high resolution structure of some medically important viruses very challenging. Here, we describe our recently employed protocols for determining the high-resolution structure of the virus-like particle of hepatitis E virus (HEV), a pathogen of viral hepatitis in human. These protocols include utilizing recombinant baculovirus system to generate sufficient amount of virus particles, single-particle cryo-EM to get an intermediate resolution structure as a phasing model, and X-ray crystallography for final atomic structure determination. Our protocols have solved the hepatitis E virus structure to the resolution of 3.5 Å. The combined methodology is generally applicable to other human infectious viruses.

Regulation of metabolism by the mediator complex

Dou Yeon Youn, Alus M. Xiaoli, Jeffrey E. Pessin, Fajun Yang

2016, 2(2-4): 69-77. doi: 10.1007/s41048-016-0031-6

Abstract(160)

PDF(148)

Abstract:
The Mediator complex was originally discovered in yeast, but it is conserved in all eukaryotes. Its bestknown function is to regulate RNA polymerase II-dependent gene transcription. Although the mechanisms by which the Mediator complex regulates transcription are often complicated by the contextdependent regulation, this transcription cofactor complex plays a pivotal role in numerous biological pathways. Biochemical, molecular, and physiological studies using cancer cell lines or model organisms have established the current paradigm of the Mediator functions. However, the physiological roles of the mammalian Mediator complex remain poorly defined, but have attracted a great interest in recent years. In this short review, we will summarize some of the reported functions of selective Mediator subunits in the regulation of metabolism. These intriguing findings suggest that the Mediator complex may be an important player in nutrient sensing and energy balance in mammals.

Crystal structures of MdfA complexed with acetylcholine and inhibitor reserpine

Ming Liu, Jie Heng, Yuan Gao, Xianping Wang

2016, 2(2-4): 78-85. doi: 10.1007/s41048-016-0028-1

Abstract(111)

PDF(156)

Abstract:
The DHA12 family of transporters contains a number of prokaryotic and eukaryote membrane proteins. Some of these proteins share conserved sites intrinsic to substrate recognition, structural stabilization and conformational changes. For this study, we chose the MdfA transporter as a model DHA12 protein to study some general characteristics of the vesicular neurotransmitter transporters (VNTs), which all belong to the DHA12 family. Two crystal structures were produced for E. coli MdfA, one in complex with acetylcholine and the other with potential reserpine, which are substrate and inhibitor of VNTs, respectively. These structures show that the binding sites of these two molecules are different. The Achbinding MfdA is mainly dependent on D34, while reserpine-binding site is more hydrophobic. Based on sequence alignment and homology modelling, we were able to provide mechanistic insights into the association between the inhibition and the conformational changes of these transporters.

Erratum to: A comprehensive procedure for antiviral inhibitor discovery using EV71 as an example