Comprehensive structural modeling and preparation of human 5-HT2A G-protein coupled receptor in functionally active form.

The serotonin 2A receptor (5-HT2A 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-HT2A R with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT2A R has marked distinction with ligand-bound conformations of 5-HT2 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-HT2A 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.

Structural modules of the stress-induced protein HflX: an outlook on its evolution and biological role.

Multifunctional proteins often show modular structures. A functional domain and the structural modules within the domain show evolutionary conservation of their spatial arrangement since that gives the protein its functionality. However, the question remains as to how members of different domains of life (Archaea, Bacteria, Eukarya), polish and perfect these modules within conserved multidomain proteins, to tailor functional proteins according to their specific requirements. In the quest for plausible answers to this question, we studied the bacterial protein HflX. HflX is a universally conserved member of the Obg-GTPase superfamily but its functional role in Archaea and Eukarya is barely known. It is a multidomain protein and possesses, in addition to its conserved GTPase domain, an ATP-binding N-terminal domain. It is involved in heat stress response in Escherichia coli and our laboratory recently identified an ATP-dependent RNA helicase activity of E. coli HflX, which is likely instrumental in rescuing ribosomes during heat stress. Because perception and response to stress is expected to be different in different life forms, the question is whether this activity is preserved in higher organisms or not. Thus, we explored the evolution pattern of different structural modules of HflX, with particular emphasis on the ATP-binding domain, to understand plausible biological role of HflX in other forms of life. Our analyses indicate that, while the evolutionary pattern of the GTPase domain follows a conserved phylogeny, conservation of the ATP-binding domain shows a complicated pattern. The limited analysis described here hints towards possible evolutionary adaptations and modifications of the domain, something which needs to be investigated in more depth in homologs from other life forms. Deciphering how nature 'tweaks' such modules, both structurally and functionally, may help in understanding the evolution of such proteins, and, on a large-scale, of stress-related proteins in general as well.

Cryo-EM structures reveal the molecular mechanism of HflX-mediated erythromycin resistance in mycobacteria.

Mycobacterial HflX confers resistance against macrolide antibiotics. However, the exact molecular mechanism is poorly understood. To gain further insights, we determined the cryo-EM structures of M. smegmatis (Msm) HflX-50S subunit and 50S subunit-erythromycin (ERY) complexes at a global resolution of approximately 3 Å. A conserved nucleotide A2286 at the gate of nascent peptide exit tunnel (NPET) adopts a swayed conformation in HflX-50S complex and interacts with a loop within the linker helical (LH) domain of MsmHflX that contains an additional 9 residues insertion. Interestingly, the swaying of this nucleotide, which is usually found in the non-swayed conformation, is induced by erythromycin binding. Furthermore, we observed that erythromycin decreases HflX's ribosome-dependent GTP hydrolysis, resulting in its enhanced binding and anti-association activity on the 50S subunit. Our findings reveal how mycobacterial HflX senses the presence of macrolides at the peptide tunnel entrance and confers antibiotic resistance in mycobacteria.

Ribosome-membrane crosstalk: Co-translational targeting pathways of proteins across membranes in prokaryotes and eukaryotes.

Ribosomes are the molecular machine of living cells designed for decoding mRNA-encoded genetic information into protein. Being sophisticated machinery, both in design and function, the ribosome not only carries out protein synthesis, but also coordinates several other ribosome-associated cellular processes. One such process is the translocation of proteins across or into the membrane depending on their secretory or membrane-associated nature. These proteins comprise a large portion of a cell's proteome and act as key factors for cellular survival as well as several crucial functional pathways. Protein transport to extra- and intra-cytosolic compartments (across the eukaryotic endoplasmic reticulum (ER) or across the prokaryotic plasma membrane) or insertion into membranes majorly occurs through an evolutionarily conserved protein-conducting channel called translocon (eukaryotic Sec61 or prokaryotic SecYEG channels). Targeting proteins to the membrane-bound translocon may occur via post-translational or co-translational modes and it is often mediated by recognition of an N-terminal signal sequence in the newly synthesizes polypeptide chain. Co-translational translocation is coupled to protein synthesis where the ribosome-nascent chain complex (RNC) itself is targeted to the translocon. Here, in the light of recent advances in structural and functional studies, we discuss our current understanding of the mechanistic models of co-translational translocation, coordinated by the actively translating ribosomes, in prokaryotes and eukaryotes.

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

The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell cultures. In this protocol, we describe a step-by-step method to express and purify serotonin 5-HT2AR using a baculoviral expression vector system in Sf9 cell cultures, derived from our work with the rat (matching Uniprot ID P14842) and human (matching Uniprot ID P28223) 5-HT2ARs. A unique feature of this method is the utilization of cell culture additives to infect cells at low multiplicity of infection, thereby using several fold less quantity of viral titer compared to prior methods without the additive. This protocol can be tweaked to selectively over-express glycosylated or non-glycosylated forms of the receptor by varying the post-infection harvest times.