G-protein coupled receptors (GPCRs), one of the largest superfamilies of cell-surface receptors, are heptahelical integral membrane proteins that play critical roles in virtually every organ system. G-protein-coupled receptors operate in membranes rich in cholesterol, with an imbalance in cholesterol level within the vicinity of GPCR transmembrane domains affecting the structure and/or function of many GPCRs, a phenomenon that has been linked to several diseases. These effects of cholesterol could result in indirect changes by altering the mechanical properties of the lipid environment or direct changes by binding to specific sites on the protein. There are a number of studies and reviews on how cholesterol modulates class A GPCRs; however, this area of study is yet to be explored for class C GPCRs, which are characterized by a large extracellular region and often form constitutive dimers. This review highlights specific sites of interaction, functions, and structural dynamics involved in the cholesterol recognition of the class C GPCRs. We summarize recent data from some typical family members to explain the effects of membrane cholesterol on the structural features and functions of class C GPCRs and speculate on their corresponding therapeutic potential.

Multidrug resistance (MDR) proteins belonging to the ATP-Binding Cassette (ABC) transporter group play a crucial role in the export of cytotoxic drugs across cell membranes. These proteins are particularly fascinating due to their ability to confer drug resistance, which subsequently leads to the failure of therapeutic interventions and hinders successful treatments. One key mechanism by which multidrug resistance (MDR) proteins carry out their transport function is through alternating access. This mechanism involves intricate conformational changes that enable the binding and transport of substrates across cellular membranes. In this extensive review, we provide an overview of ABC transporters, including their classifications and structural similarities. We focus specifically on well-known mammalian multidrug resistance proteins such as MRP1 and Pgp (MDR1), as well as bacterial counterparts such as Sav1866 and lipid flippase MsbA. By exploring the structural and functional features of these MDR proteins, we shed light on the roles of their nucleotide-binding domains (NBDs) and transmembrane domains (TMDs) in the transport process. Notably, while the structures of NBDs in prokaryotic ABC proteins, such as Sav1866, MsbA, and mammalian Pgp, are identical, MRP1 exhibits distinct characteristics in its NBDs. Our review also emphasizes the importance of two ATP molecules for the formation of an interface between the two binding sites of NBD domains across all these transporters. ATP hydrolysis occurs following substrate transport and is vital for recycling the transporters in subsequent cycles of substrate transportation. Specifically, among the studied transporters, only NBD2 in MRP1 possesses the ability to hydrolyze ATP, while both NBDs of Pgp, Sav1866, and MsbA are capable of carrying out this reaction. Furthermore, we highlight recent advancements in the study of MDR proteins and the alternating access mechanism. We discuss the experimental and computational approaches utilized to investigate the structure and dynamics of MDR proteins, providing valuable insights into their conformational changes and substrate transport. This review not only contributes to an enhanced understanding of multidrug resistance proteins but also holds immense potential for guiding future research and facilitating the development of effective strategies to overcome multidrug resistance, thus improving therapeutic interventions.

The surface protein hemagglutinin (HA) of the influenza virus plays a pivotal role in facilitating viral infection by binding to sialic acid receptors on host cells. Its conformational state is pH-sensitive, impacting its receptor-binding ability and evasion of the host immune response. In this study, we conducted extensive equilibrium microsecond-level all-atom molecular dynamics (MD) simulations of the HA protein to explore the influence of low pH on its conformational dynamics. Specifically, we investigated the impact of protonation on conserved histidine residues (H106₂) located in the hinge region of HA2. Our analysis encompassed comparisons between nonprotonated (NP), partially protonated (1P, 2P), and fully protonated (3P) conditions. Our findings reveal substantial pH-dependent conformational alterations in the HA protein, affecting its receptor-binding capability and immune evasion potential. Notably, the nonprotonated form exhibits greater stability compared to protonated states. Conformational shifts in the central helices of HA2 involve outward movement, counterclockwise rotation of protonated helices, and fusion peptide release in protonated systems. Disruption of hydrogen bonds between the fusion peptide and central helices of HA2 drives this release. Moreover, HA1 separation is more likely in the fully protonated system (3P) compared to nonprotonated systems (NP), underscoring the influence of protonation. These insights shed light on influenza virus infection mechanisms and may inform the development of novel antiviral drugs targeting HA protein and pH-responsive drug delivery systems for influenza.

YidC is a membrane protein that plays an important role in inserting newly generated proteins into lipid membranes. The Sec-dependent complex is responsible for inserting proteins into the lipid bilayer in bacteria. YidC facilitates the insertion and folding of membrane proteins, both in conjunction with the Sec complex and independently. Additionally, YidC acts as a chaperone during the folding of proteins. Multiple investigations have conclusively shown that Gram-positive bacterial YidC has Sec-independent insertion mechanisms. Through the use of microsecond-level all-atom molecular dynamics (MD) simulations, we have carried out an in-depth investigation of the YidC protein originating from Gram-negative bacteria. This research sheds light on the significance of multiple domains of the YidC structure at a detailed molecular level by utilizing equilibrium MD simulations. Specifically, multiple models of YidC embedded in the lipid bilayer were constructed to characterize the critical role of the C2 loop and the periplasmic domain (PD) present in Gram-negative YidC, which is absent in its Gram-positive counterpart. Based on our results, the C2 loop plays a role in the overall stabilization of the protein, most notably in the transmembrane (TM) region, and it also has an allosteric influence on the PD region. We have found critical inter- and intradomain interactions that contribute to the stability of the protein and its function. Finally, our study provides a hypothetical Sec-independent insertion mechanism for Gram-negative bacterial YidC.

G-protein coupled receptors (GPCRs), one of the largest superfamilies of cell-surface receptors, are heptahelical integral membrane proteins that play critical roles in virtually every organ system. G-protein-coupled receptors operate in membranes rich in cholesterol, with an imbalance in cholesterol level within the vicinity of GPCR transmembrane domains affecting the structure and/or function of many GPCRs, a phenomenon that has been linked to several diseases. These effects of cholesterol could result in indirect changes by altering the mechanical properties of the lipid environment or direct changes by binding to specific sites on the protein. There are a number of studies and reviews on how cholesterol modulates class A GPCRs, however, this area of study is yet to be explored for class C GPCRs, which are characterized by a large extracellular region and often form constitutive dimers. This review highlights specific sites of interaction, functions, and structural dynamics involved in the cholesterol recognition of the class C GPCRs. We summarize recent data from some typical family members to explain the effects of membrane cholesterol on the structural features and functions of Class C GPCRs and speculate on their corresponding therapeutic potential.

Archaeal group II chaperonins, also known as heat shock proteins (HSPs), are abundantly expressed in Sulfolobales. HSPα and HSPβ gene expression is upregulated during thermal shock. HSPs form large 18-mer complexes that assist in folding nascent proteins and protecting resident proteins during thermal stress. Engineered HSPs have been designed for industrial applications. Since temperature flux in the geothermal habitats of Sulfolobales impacts intracellular temperature, it follows that HSPs have developed thermotolerance. However, despite the low pH (i.e., pH < 4) typical for these habitats, intracellular pH in Sulfolobales is maintained at ~6.5. Therefore, it is not presumed that HSPs have evolved acid-tolerance. To test tolerance to low pH, HSPs were studied at various pH and temperature values. Both circular dichroism and intrinsic fluorescence indicate that HSPα and HSPβ retain structural integrity at neutral pH over a wide range of temperatures. Structural integrity is compromised for all HSPs at ultra-low pH (e.g., pH 2). Secondary structures in HSPs are resilient under mildly acidic conditions (pH 4) but Anilino naphthalene 8-sulfonate binding shows shifts in tertiary structure at lower pH. Trypsin digestion shows that the HSPβ-coh backbone is the most flexible and HSPβ is the most resilient. Overall, results suggest that HSPα and HSPβ exhibit greater thermostability than HSPβ-coh and that there are limits to HSP acid-tolerance. Molecular dynamics (MD) simulations complement the wet lab data. Specifically, MD suggests that the HSPβ secondary structure is the most stable. Also, despite similarities in pH- and temperature-dependent behavior, there are clear differences in how each HSP subtype is perturbed.