Proton Coupling and the Multiscale Kinetic Mechanism of a Peptide Transporter

ABSTRACTThe proton electrochemical gradient drives substrate transport across the cell membrane via a diverse set of secondary active transporters. Proton coupled peptide transporters (POTs) are important for peptide transport in prokaryotes and eukaryotic cells, where they mediate the uptake of di- and tri-peptides in addition to drug and pro-drug molecules. Previously, we captured a POT transporter from Staphylococcus hominis, PepTSh, in a cytoplasm-facing, inward open state (Minhas et al., 2018). Biochemical experiments have further revealed several critical residues for proton coupled transport; however, the precise role played by these residues in coupling proton binding to conformational changes as well as the timescales for proton transfers have remained obscure. Here, we employed multiscale modeling, including classical molecular dynamics, reactive molecular dynamics, and enhanced free energy sampling to characterize proton coupling within this transporter. We show directly that proton binding to a glutamate on TM7 opens the extracellular gate. The inward proton flow is found to induce movement of the peptide towards the cytosol by varying the protonation state of a second conserved glutamate on TM10. We also show that proton movement between TM7 and TM10 is thermodynamically driven and kinetically permissible, revealing a mechanism for proton movement inside the transporter.

HIFs: New arginine mimic inhibitors of the Hv1 channel with improved VSD–ligand interactions

The human voltage-gated proton channel Hv1 is a drug target for cancer, ischemic stroke, and neuroinflammation. It resides on the plasma membrane and endocytic compartments of a variety of cell types, where it mediates outward proton movement and regulates the activity of NOX enzymes. Its voltage-sensing domain (VSD) contains a gated and proton-selective conduction pathway, which can be blocked by aromatic guanidine derivatives such as 2-guanidinobenzimidazole (2GBI). Mutation of Hv1 residue F150 to alanine (F150A) was previously found to increase 2GBI apparent binding affinity more than two orders of magnitude. Here, we explore the contribution of aromatic interactions between the inhibitor and the channel in the presence and absence of the F150A mutation, using a combination of electrophysiological recordings, classic mutagenesis, and site-specific incorporation of fluorinated phenylalanines via nonsense suppression methodology. Our data suggest that the increase in apparent binding affinity is due to a rearrangement of the binding site allowed by the smaller residue at position 150. We used this information to design new arginine mimics with improved affinity for the nonrearranged binding site of the wild-type channel. The new compounds, named “Hv1 Inhibitor Flexibles” (HIFs), consist of two “prongs,” an aminoimidazole ring, and an aromatic group connected by extended flexible linkers. Some HIF compounds display inhibitory properties that are superior to those of 2GBI, thus providing a promising scaffold for further development of high-affinity Hv1 inhibitors.

System of electron-proton. Movement, rotation, pulsation and Gravity Forces

The new Field Theory consist two new Axioms and eight new Laws. It has been proposed and developed in previous reports by the same author. This report uses two axioms and six laws only. According to the first axiom (Axiom1), the author replaces uniform motion in a closed circle with non-uniform motion in an open vortex. According to the second axiom (Axiom2), it exists a pairs of vortices that are mutually orthogonal or they work in a system of resonance. The most probable of all of variants is the following pair: accelerating vortex from the center outwards connected with a decelerating vortex from the periphery inwards. This case is a model of the connected proton-electron pair. In this report the properties of a system only of linked electrons and protons are studied. It is known that the Electromagnetic Field propagates at a constant speed and the waves are only transverse. According to the new Axioms and Laws in the electron-proton system, the internal connections are of variable speed and the waves are not only transverse but and longitudinal. It appears that the interactions between the proton and the electron are not Electromagnetic. They include cross vortex with variable velocity and longitudinal vortex with variable velocity as well. From previous developments it is clear that the electron is not a concentric open vortex but an eccentric open vortex, centered in the second quadrant. And the proton is not a concentric open vortex but an eccentric open vortex, centered in the first quadrant. This is the reason for the formation of eccentricity vectors that decompose along the x and y axes. Because the eccentricity of the electron is greater than the eccentricity of the proton then the component along they axis rotates the electron around the proton (in orbit). And besides, since the decelerating vortex of the electron emits elementary decelerating vortices (Law 5) inward which are bent in the direction of the decelerating velocity, the electron will rotate parasitically and slowly around its own axis (in spin). The electron and proton are repelled gravitationally by a transverse component and are attracted gravitationally by a longitudinal component which are with variable speed. The existence of feedback between the electron and the proton (Law 7 and Law 8) explains the reason for the presence of elementary cross vortices. When they are emitted outward - are called “free energy”. And because they are invisible -are called and “black matter” as well.

Bio-inspired protonic memristor devices based on metal complexes with proton-coupled electron transfer

A new type of memristor inspired by bio-membranes is presented, based on the proton movement resulting from proton-coupled electron transfer (PCET) processes in dinuclear Ru complexes, whereby a two-terminal device based on said Ru complexes and a proton-conducting polymer was constructed as a proof-of-concept.

A designed heme-[4Fe-4S] metalloenzyme catalyzes sulfite reduction like the native enzyme

Multielectron redox reactions often require multicofactor metalloenzymes to facilitate coupled electron and proton movement, but it is challenging to design artificial enzymes to catalyze these important reactions, owing to their structural and functional complexity. We report a designed heteronuclear heme-[4Fe-4S] cofactor in cytochromecperoxidase as a structural and functional model of the enzyme sulfite reductase. The initial model exhibits spectroscopic and ligand-binding properties of the native enzyme, and sulfite reduction activity was improved—through rational tuning of the secondary sphere interactions around the [4Fe-4S] and the substrate-binding sites—to be close to that of the native enzyme. By offering insight into the requirements for a demanding six-electron, seven-proton reaction that has so far eluded synthetic catalysts, this study provides strategies for designing highly functional multicofactor artificial enzymes.

Proton movement and coupling in the POT family of peptide transporters

POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.

Getting the chemistry right: protonation, tautomers and the importance of H atoms in biological chemistry

There are more H atoms than any other type of atom in an X-ray crystal structure of a protein–ligand complex, but as H atoms only have one electron they diffract X-rays weakly and are `hard to see'. The positions of many H atoms can be inferred by our chemical knowledge, and such H atoms can be added with confidence in `riding positions'. For some chemical groups, however, there is more ambiguity over the possible hydrogen placements, for example hydroxyls and groups that can exist in multiple protonation states or tautomeric forms. This ambiguity is far from rare, since about 25% of drugs have more than one tautomeric form. This paper focuses on the most common, `prototropic', tautomers, which are isomers that readily interconvert by the exchange of an H atom accompanied by the switch of a single and an adjacent double bond. Hydrogen-exchange rates and different protonation states of compounds (e.g. buffers) are also briefly discussed. The difference in heavy (non-H) atom positions between two tautomers can be small, and careful refinement of all possible tautomers may single out the likely bound ligand tautomer. Experimental methods to determine H-atom positions, such as neutron crystallography, are often technically challenging. Therefore, chemical knowledge and computational approaches are frequently used in conjugation with experimental data to deduce the bound tautomer state. Proton movement is a key feature of many enzymatic reactions, so understanding the orchestration of hydrogen/proton motion is of critical importance to biological chemistry. For example, structural studies have suggested that, just as a chemist may use heat, some enzymes use directional movement to protonate specific O atoms on phosphates to catalyse phosphotransferase reactions. To inhibit `wriggly' enzymes that use movement to effect catalysis, it may be advantageous to have inhibitors that can maintain favourable contacts by adopting different tautomers as the enzyme `wriggles'.