Coxsackievirus B3 protease 3C: expression, purification, crystallization and preliminary structural insights

Abstract

Viral proteases are proteolytic enzymes that orchestrate the assembly of viral components during the viral life cycle and proliferation. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis are presented of protease 3C, the main protease of an emerging enterovirus, coxsackievirus B3, that is responsible for many cases of viral myocarditis. Polycrystalline protein precipitates suitable for X-ray powder diffraction (XRPD) measurements were produced in the presence of 22-28%(w/v) PEG 4000, 0.1 M Tris-HCl, 0.2 M MgCl in a pH range from 7.0 to 8.5. A polymorph of monoclinic symmetry (space group C2, unit-cell parameters a = 77.9, b = 65.7, c = 40.6 Å, β = 115.9°) was identified via XRPD. These results are the first step towards the complete structural determination of the molecule via XRPD and a parallel demonstration of the accuracy of the method.

Applications of X-ray Powder Diffraction in Protein Crystallography and Drug Screening

“…Using laboratory instrumentation (Malvern Panalytical, X' Pert PRO), initial extraction of unit cell parameters and crystal symmetry (indexing) was feasible, while the best diffraction profiles in terms of angular and d-spacing resolution were obtained at the ESRF, allowing accurate identification of unit-cell parameters and characterization of peak shape and background coefficients in the absence of a structural model using Pawley method [75]. XRPD data analysis demonstrated no structural modifications or alterations in the diffraction peak positions throughout the crystallization conditions examined, with all samples containing crystals of monoclinic symmetry (space group C2) ( Figure 3) [71].…”

“…In addition, the application of the XRPD method provides the advantage of considerably reducing the amount of time necessary for fine-tuning crystallization experiments, and in the case of virus proteins, it may be useful to examine multiple crystallization conditions by investigating the formation of different crystalline polymorphs [70][71][72]. This allows for the examination of physicochemical characteristics that each polymorph bears, as well as the ability to bind molecules that inactivate the action of any such protein, considering their potential utility as drug precursors.…”

“…The applicability of XRPD measurements in antiviral research, and its ability to provide preliminary structural information as first shown from Papageorgiou and colleagues (2010), triggered the research around difficult-to-crystallize virus proteins. Recently, a study focused on a 20.5kDa protein, protease 3C (3C pro ) of an emerging Enterovirus, Coxsackievirus B3 (CVB3), has come to support this claim further [71]. CVB3 may cause various diseases ranging from pleuropneumonia or "Bornholme disease" to myocarditis leading to permanent heart damage or even death [73], while this molecule is comprised of the functional virus proteins and is responsible for the majority of proteolytic cleavages occurring within the host cell [74].…”

“…Crystals 2020, 10, 54 6 of 34 modifications or alterations in the diffraction peak positions throughout the crystallization conditions examined, with all samples containing crystals of monoclinic symmetry (space group C2) ( Figure 3) [71]. Recent measurements conducted at the Swiss Light Source (SLS) allowed for the collection of data of improved d-spacing resolution.…”

“…The black, red and blue lines represent the experimental data, the calculated pattern and the difference between the experimental and caculated profiles, respectively. The vertical bars correspond to Bragg reflections compatible with space group C2 [71].…”