Comprehensive structural modeling and preparation of human 5‐HT_2A G‐protein coupled receptor in functionally active form

Abstract

The serotonin 2A receptor (5-HT 2A R) is an important member of the G-protein coupled receptor (GPCR) family involved in an array of neuromodulatory functions. Although the high-resolution structures of truncated versions of GPCRs, captured in ligand-bound conformational states, are available, the structures lack several functional regions, which have crucial roles in receptor response. Here, in order to understand the structure and dynamics of the ligand-free form of the receptor, we have performed meticulous modeling of the 5-HT 2A R with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT 2A R has marked distinction with ligand-bound conformations of 5-HT 2 subfamily proteins and exhibits extensive backbone flexibility across the loop regions, suggesting the importance of purifying the receptor in its native form for further studies. Hence, we have standardized a strategy that efficiently increases the expression of 5-HT 2A R by infecting Sf9 cells with a very low multiplicity of infection of baculovirus in conjunction with production boost additive and subsequently, purify the full-length receptor. Furthermore, we have optimized the selective over-expression of glycosylated and nonglycosylated forms of the receptor merely by switching the postinfection growth time, a method that has not been reported earlier. K E Y W O R D S 5-HT 2A GPCR, baculoviral expression system, homology modeling, molecular dynamics simulation, protein over-expression and purification

Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock

“…Hence, it may be prudent to optimize these factors, especially if it is more economical to expend baculoviral stock and save PBA, or vice versa. Moreover, as noted previously (Mozumder et al, 2020), the extent of glycosylation in cultures grown with PBA can be modulated by varying the post-infection harvest time. Hence the time of harvest may also be optimized to select the desirable amount of glycosylation, by running Western blot analysis of cell lysate from cultures grown over a range of post-infection harvest times (see for suitable buffers for Western blotting).…”

Preferential Coupling of Dopamine D2S and D2L Receptor Isoforms with Gi1 and Gi2 Proteins—In Silico Study

“…However, intracellular loops, in particular ICL3, play an important role in receptor activation due to their interaction with G proteins and the influence on G protein preferential coupling [32][33][34]. Recently, a full model of human serotonin 5-HT 2A receptor has been reported as an example of this challenging loop modeling [15].…”

“…In spite of about 350 GPCR crystal structures including 85 active structures [14] in the Protein Data Bank, the full experimentally solved structures of receptors with long and flexible ICL3 are not yet available. Due to limitations of experimental methods, at present, such structures can be only obtained using molecular modeling approaches as it has been recently done, e.g., for the human serotonin 5-HT 2A receptor [15]. In this work, we aimed to construct complete models of D 2S and D 2L isoforms in complex with dopamine and both G i1 and G i2 proteins and, subsequently, to examine possible G protein coupling preferences of these isoforms and the receptor activation/deactivation hallmarks upon agonist binding and G protein coupling with molecular dynamics (MD) techniques.…”

A qualitative discussion on the principle of similar supporting the use of Arsenicum album against corona infectious diseases

“…It would be worthwhile to mention at this point that the probability of relation between two separate systems was discussed mathematically by Schrodinger [34,35] . If there is a common observable in the two systems (in our case the sin/ion transfer between NAM and the diseased cell and between NAM and medicinal molecules to get the signature of the medicinal molecules) and when such two observable are related coherently to some of their states of the two systems, by some definite common observable (ions/sins transfer in both the MAM and DC systems) one to one correspondence persists as time advances and the observables of the first system (say NAM) change their ions/electrons in the way that the observables in the second system (say in the DC) undergo a certain contacttransformation [36,37] . Definitely the situation is complex executing a new field of research, called Quantum Biology [38,41] (where physics meets biology), which is still in the developing state.…”