Molecular mechanism of thiamine pyrophosphate import into mitochondria: a molecular simulation study.

The import of thiamine pyrophosphate (TPP) through both mitochondrial membranes was studied using a total of 3-µs molecular dynamics simulations. Regarding the translocation through the mitochondrial outer membrane, our simulations support the conjecture that TPP uses the voltage-dependent anion channel, the major pore of this membrane, for its passage to the intermembrane space, as its transport presents significant analogies with that used by other metabolites previously studied, in particular with ATP. As far as passing through the mitochondrial inner membrane is concerned, our simulations show that the specific carrier of TPP has a single binding site that becomes accessible, through an alternating access mechanism. The preference of this transporter for TPP can be rationalized mainly by three residues located in the binding site that differ from those identified in the ATP/ADP carrier, the most studied member of the mitochondrial carrier family. The simulated transport mechanism of TPP highlights the essential role, at the energetic level, of the contributions coming from the formation and breakage of two networks of salt bridges, one on the side of the matrix and the other on the side of the intermembrane space, as well as the interactions, mainly of an ionic nature, formed by TPP upon its binding. The energy contribution provided by the cytosolic network establishes a lower barrier than that of the matrix network, which can be explained by the lower interaction energy of TPP on the matrix side or possibly a uniport activity.

Lipid composition and salt concentration as regulatory factors of the anion selectivity of VDAC studied by coarse-grained molecular dynamics simulations.

The voltage-dependent anion channel (VDAC) is a mitochondrial outer membrane protein whose fundamental function is to facilitate and regulate the flow of metabolites between the cytosol and the mitochondrial intermembrane space. Using coarse-grained molecular dynamics simulations, we investigated the dependence of VDAC selectivity towards small inorganic anions on two factors: the ionic strength and the lipid composition. In agreement with experimental data we found that VDAC becomes less anion selective with increasing salt concentration due to the screening of a few basic residues that point into the pore lumen. The molecular dynamics simulations provide insight into the regulation mechanism of VDAC selectivity by the composition in the lipid membrane and suggest that the ion distribution is differently modulated by POPE compared to the POPC bilayer. This occurs through the more persistent interactions of acidic residues located at both rims of the ß-barrel with head groups of POPE which in turn impact the electrostatic potential and thereby the selectivity of the pore. This mechanism occurs not only in POPE single component membranes but also in a mixed POPE/POPC bilayer by an enrichment of POPE over POPC lipids on the surface of VDAC. Thus we show here that computationally-inexpensive coarse-grained simulations are able to capture, in a semi-quantitative way, essential features of VDAC anion selectivity and could pave the way toward a molecular level understanding of metabolite transport in natural membranes.

Balance between Coiled-Coil Stability and Dynamics Regulates Activity of BvgS Sensor Kinase in Bordetella.

UNLABELLED: The two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS) domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil's dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases. IMPORTANCE: The two-component system BvgAS of the whooping cough agent Bordetella pertussis regulates the virulence factors necessary for infection in a coordinated manner. BvgS is the prototype of a family of sensor kinase proteins found in major bacterial pathogens. When BvgS functions as a kinase, B. pertussis is virulent, and the bacterium shifts to an avirulent phase after BvgS senses chemicals that make it switch to phosphatase. Our goal is to decipher the signaling mechanisms of BvgS in order to understand virulence regulation in Bordetella, which may lead to new antimicrobial treatments targeting those two-component systems. We discovered that the activity of BvgS is regulated in a mechanical manner. A short region of the protein that precedes the enzymatic domain switches between two states in response to signal perception by other BvgS domains. This switch region is conserved among BvgS homologs, and thus, the regulation uncovered here will likely be relevant for the family.