Homology Modeling and Molecular Dynamics Simulation Combined with X-ray Solution Scattering Defining Protein Structures of Thromboxane and Prostacyclin Synthases

Abstract

A combination of molecular dynamics (MD) simulations and X-ray scattering (SAXS) has emerged as the approach of choice for studying protein structures and dynamics in solution. This approach has potential applications for membrane proteins that neither are soluble nor form crystals easily. We explore the water-coupled dynamic structures of thromboxane synthase (TXAS) and prostacyclin synthase (PGIS) from scanning HPLC-SAXS measurements combined with MD ensemble analyses. Both proteins are heme-containing enzymes in the cytochrome P450 family, known as prostaglandin H (PGH) isomerase, with counter-functions in regulation of platelet aggregation. Currently, the X-ray crystallographic structures of PGIS are available, but those for TXAS are not. The use of homology modeling of the TXAS structure with ns-μs explicit water solvation MD simulations allows much more accurate estimation of the configuration space with loop motion and origin of the protein behaviors in solution. In contrast to the stability of the conserved PGIS structure in solution, the pronounced TXAS flexibility has been revealed to have unstructured loop regions in connection with the characteristic P450 structural elements. The MD-derived and experimental-solution SAXS results are in excellent agreement. The significant protein internal motions, whole-molecule structures, and potential problems with protein folding, crystallization, and functionality are examined.

Impact of peripheral mutations on the access channels of human cytochrome P450 1A2

“…In the wild type CYP1A2, the water channel consists of the loop region between B and C helices (BC loop, Figure 5A ). Small-angle X-ray scattering (SAXS) experiments and molecular dynamics simulations indicate that the BC loop is one of the membrane-interacting regions, and CYP1A2 has the most contact area for the BC loop among all the CYP proteins (Berka et al, 2013; Skar-Gislinge et al, 2015; Yang et al, 2017).…”

Molecular dynamics simulation combined with small‐angle X‐ray/neutron scattering defining solution‐state protein structures

“…Here we choose two antagonistic proteins, PGIS and TXAS, for demonstration. [ 11 ] Currently, the X‐ray crystallographic structures of PGIS are available, but lack of TXAS structures. Given in Figure 2a, we used an online size exclusion high‐performance liquid chromatographic (SE‐HPLC) system to collect individual monodisperse from oligomer.…”

“…[ 7–10 ] To shed light on how the chemical constitution of the protein, the water interactions, and the dynamics of its structure underlie the specific mechanisms of enzyme functions, in this paper, we review an integrated approach using small‐angle X‐ray and neutron scattering (SAXS and SANS) in combination with molecular dynamics (MD) simulation (Figure 1 ) to investigate the structural dynamics of a protein, thus shedding light on its function. [ 11,12 ]…”

“…The findings of the study indicate that the scanning HPLC−SAXS approach effectively excludes scattering containment from coexisting oligomer species while collecting mono‐dispersion monomer protein structures (see Figure 1c) and limits damage from radiation via an on‐line flow system. [ 11,17,18 ]…”

“…We demonstrated an approach combining simulations of all‐atom molecular dynamics and small‐angle X‐ray scattering (SAXS)/ultraviolet–visible spectroscopy (UV–vis) absorption measurements, which allows probing of the structures and dynamical behaviors of two counter‐functioning proteins, thromboxane synthase (TXAS) and prostacyclin synthase (PGIS), in solution. [ 11 ] A large number of MD structures can be used to simulate SAXS and SANS profiles (Figure 1b ) in the software CRYSOL and CRYSON. [ 19,20 ] The results of MD and experimental‐solution SAXS had excellent agreement.…”