The Influenza Matrix 2 (M2) protein is the target of Amantadine and Rimantadine which block its H(+) channel activity. However, the potential of these aminoadamantyls to serve as anti-flu agents is marred by the rapid resistance that the virus develops against them. Herein, using a cell based assay that we developed, we identify two new aminoadamantyl derivatives that show increased activity against otherwise resistant M2 variants. In order to understand the distinguishing binding patterns of the different blockers, we computed the potential of mean force of the drug binding process. The results reveal that the new derivatives are less mobile and bind to a larger pocket in the channel. Finally, such analyses may prove useful in designing new, more effective M2 blockers as a means of curbing influenza. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.
G-protein–coupled receptors (GPCRs) are a large group of membrane-bound receptor proteins that are involved in a plethora of diverse processes (e.g., vision, hormone response). In mammals, and particularly in humans, GPCRs are involved in many signal transduction pathways and, as such, are heavily studied for their immense pharmaceutical potential. Indeed, a large fraction of drugs target various GPCRs, and drug-development is often aimed at GPCRs. Therefore, understanding the activation of GPCRs is a challenge of major importance both from fundamental and practical considerations. And yet, despite the remarkable progress in structural understanding, we still do not have a translation of the structural information to an energy-based picture. Here we use coarse-grained (CG) modeling to chart the free-energy landscape of the activation process of the β-2 adrenergic receptor (β2AR) as a representative GPCR. The landscape provides the needed tool for analyzing the processes that lead to activation of the receptor upon binding of the ligand (adrenaline) while limiting constitutive activation. Our results pave the way to better understand the biological mechanisms of action of the β2AR and GPCRs, from a physical chemistry point of view rather than simply by observing the receptor’s behavior physiologically.
The nature of the action of voltage-activated proton transport proteins is a conundrum of great current interest. Here we approach this issue by exploring the action of Hv1, a voltage-gated proton channel found in different cells in humans and other organisms. Our study focuses on evaluating the free energy of transporting a proton through the channel, as well as the effect of the proton transfer through D112, in both the closed and open channel conformations. It is found that D112 allows a transported proton to bypass the electrostatic barrier of the open channel, while not being able to help in passing the barrier in the closed form. This reflects the change in position of the gating arginine residues relative to D112, upon voltage activation. Significantly, the effect of D112 accounts for the observed trend in selectivity by overcoming the electrostatic barrier at its highest point. Thus, the calculations provide a structure/function correlation for the Hv1 system. The present work also clarifies that the action of Hv1 is not controlled by a Grotthuss mechanism but, as is always the case, by the protein electrostatic potential at the rate-limiting barriers.
Zinc is a vital trace element crucial for the proper function of some 3,000 cellular proteins. Specifically, zinc is essential for key physiological processes including nucleic acid metabolism, regulation of gene expression, signal transduction, cell division, immune- and nervous system functions, wound healing, and apoptosis. Consequently, impairment of zinc homeostasis disrupts key cellular functions resulting in various human pathologies. Mammalian zinc transport proceeds via two transporter families ZnT and ZIP. However, the detailed mechanism of action of ZnT2, which is responsible for vesicular zinc accumulation and zinc secretion into breast milk during lactation, is currently unknown. Moreover, although the putative coupling of zinc transport to the proton gradient in acidic vesicles has been suggested, it has not been conclusively established. Herein we modeled the mechanism of action of ZnT2 and demonstrated both computationally and experimentally, using functional zinc transport assays, that ZnT2 is indeed a proton-coupled zinc antiporter. Bafilomycin A1, a specific inhibitor of vacuolar-type proton ATPase (V-ATPase) which alkalizes acidic vesicles, abolished ZnT2-dependent zinc transport into intracellular vesicles. Moreover, using LysoTracker Red and Lyso-pHluorin, we further showed that upon transient ZnT2 overexpression in intracellular vesicles and addition of exogenous zinc, the vesicular pH underwent alkalization, presumably due to a proton-zinc antiport; this phenomenon was reversed in the presence of TPEN, a specific zinc chelator. Finally, based on computational energy calculations, we propose that ZnT2 functions as an antiporter with a stoichiometry of 2H + /Zn 2+ ion. Hence, ZnT2 is a proton motive force-driven, electroneutral vesicular zinc exchanger, concentrating zinc in acidic vesicles on the expense of proton extrusion to the cytoplasm.
The molecular basis of the function of transporters is a problem of significant importance, and the emerging structural information has not yet been converted to a full understanding of the corresponding function. This work explores the molecular origin of the function of the bacterial Na + /H + antiporter NhaA by evaluating the energetics of the Na + and H + movement and then using the resulting landscape in Monte Carlo simulations that examine two transport models and explore which model can reproduce the relevant experimental results. The simulations reproduce the observed transport features by a relatively simple model that relates the protein structure to its transporting function. Focusing on the two key aspartic acid residues of NhaA, D163 and D164, shows that the fully charged state acts as an Na + trap and that the fully protonated one poses an energetic barrier that blocks the transport of Na + . By alternating between the former and latter states, mediated by the partially protonated protein, protons, and Na + can be exchanged across the membrane at 2:1 stoichiometry. Our study provides a numerical validation of the need of large conformational changes for effective transport. Furthermore, we also yield a reasonable explanation for the observation that some mammalian transporters have 1:1 stoichiometry. The present coarse-grained model can provide a general way for exploring the function of transporters on a molecular level.transport | computational biology | membrane protein
The detailed dynamics of the cycle of myosin-V are explored by simulation approaches, examining the nature of the energy-driven motion. Our study started with Langevin dynamics (LD) simulations on a very coarse landscape with a single rate-limiting barrier and reproduced the stall force and the hand-over-hand dynamics. We then considered a more realistic landscape and used timedependent Monte Carlo (MC) simulations that allowed trajectories long enough to reproduce the force/velocity characteristic sigmoidal correlation, while also reproducing the hand-over-hand motion. Overall, our study indicated that the notion of a downhill lever-up to lever-down process (popularly known as the powerstroke mechanism) is the result of the energetics of the complete myosin-V cycle and is not the source of directional motion or force generation on its own. The present work further emphasizes the need to use well-defined energy landscapes in studying molecular motors in general and myosin in particular.M yosin constitutes a superfamily of molecular motors comprising both the nonprocessive single-headed motors that are efficient in generating force on the actin filaments (e.g., myosin-II) and highly processive double-headed motors that can transport cellular load using tracks laid out by the cytoskeletal actin filaments (e.g., myosin-V, myosin-VI) (1, 2). The generation of force and unidirectional motion in each of the myosin molecules predominantly comprises tightly coupled events involving (i) binding and hydrolysis of ATP, followed by release of the products ADP and inorganic phosphate (P i ), occurring at the nucleotide-binding domain; (ii) binding and release of actin through the actin-binding domain; and (iii) a large conformational change of the lever arm that generates mechanical motion. Although the basic mechanochemical cycle is conserved in all members of the myosin superfamily, the exact nature of the coupling between the above steps in myosins, which decides the force-generating and load-bearing characteristics of different members, is unknown (SI Background).Numerous experiments, including kinetics and thermodynamic studies (3), high-resolution structural studies (4), electron microscopy studies (5), atomic force microscopy (AFM), and singlemolecule experiments (2, 6, 7), have advanced our understanding of the action of the system. Although details about the dynamical nature of the myosin-V, along with a quantitative knowledge of the force-response, have been learned from single-molecule studies, the structural studies have provided us with an atomistic knowledge of the key conformational states involved during the cycle, namely, the lever-up (pre) and lever-down (post) states (Fig. S1). In addition, crucial information has been gained on the kinetics of the chemical and ligand-binding/release steps that make up the complete cycle. Based on this experimental information, several theoretical modeling studies (8-17) explored the mechanochemical cycle, mostly using phenomenological modeling approaches, accompanied by s...
Multiscale modeling provides a very powerful means of studying complex biological systems. An important component of this strategy involves coarse-grained (CG) simplifications of regions of the system, which allow effective exploration of complex systems. Here we studied aspects of CG modeling of the human zinc transporter ZnT2. Zinc is an essential trace element with 10% of the proteins in the human proteome capable of zinc binding. Thus, zinc deficiency or impairment of zinc homeostasis disrupt key cellular functions. Mammalian zinc transport proceeds via two transporter families: ZnT and ZIP; however, little is known about the zinc permeation pathway through these transporters. As a step towards this end, we herein undertook comprehensive computational analyses employing multiscale techniques, focusing on the human zinc transporter ZnT2 and its bacterial homologue, YiiP. Energy calculations revealed a favorable pathway for zinc translocation via alternating access. We then identified key residues presumably involved in the passage of zinc ions through ZnT2 and YiiP, and functionally validated their role in zinc transport using site-directed mutagenesis of ZnT2 residues. Finally, we use a CG Monte Carlo simulation approach to sample the transition between the inward-facing and the outward-facing states. We present our structural models of the inward- and outward-facing conformations of ZnT2 as a blueprint prototype of the transporter conformations, including the putative permeation pathway and participating residues. The insights gained from this study may facilitate the delineation of the pathways of other zinc transporters, laying the foundations for the molecular basis underlying ion permeation. This may possibly facilitate the development of therapeutic interventions in pathological states associated with zinc deficiency and other disorders based on loss-of-function mutations in solute carriers.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.