Biology subjects

Forrest, L. R.

Publications and source records attributed to Forrest, L. R..

3 recordsLinked to original sources

bioRxiv2018

EncoMPASS: an Encyclopedia of Membrane Proteins Analyzed by Structure and Symmetry

Protein structure determination and prediction, active site detection, and protein sequence alignment techniques all exploit information about protein structure and structural relationships. For membrane proteins, however, there is no agreement among available online tools for highlighting and mapping such structural similarities. Moreover, no available resource provides a systematic overview of quaternary and internal symmetries, and their orientation with respect to the membrane, despite the fact that these properties can provide key insights into membrane protein function. To address these issues, we created the Encyclopedia of Membrane Proteins Analyzed by Structure and Symmetry (EncoMPASS), a database for relating integral membrane proteins of known structure from the points of view of sequence, structure, and symmetry. EncoMPASS is accessible at https://encompass.ninds.nih.gov and its contents can be easily downloaded. This allows the user not only to focus on specific systems, but also to study general properties of the structure and evolution of membrane proteins.\n\nHighlights-EncoMPASS relates and analyzes known structures of membrane proteins\n-Structure and sequence similarity is assessed through alignments and topology considerations, not clustering\n-Symmetry is detected based on CE-Symm and SymD using a multi-step procedure

bioinformatics↗

bioRxiv2018

Assignment of the resting state of the secondary active transporter BetP by integrating spectroscopic measurements and molecular simulations

The glycine betaine symporter BetP regulates the osmotic stress response of Corynebacterium glutamicum, a soil bacterium used extensively in biotechnology. Although BetP is a homotrimer, biochemical studies have shown that each protomer is able to transport its substrate independently. Crystallographic structures of BetP have been determined in several conformations, seemingly capturing outward-open, inward-open and occluded states, both loaded with the substrate and in the apo form. However, it has been challenging to establish a correspondence between each of these structures and specific states in the mechanism of the transporter under more physiological conditions. To this end, we examined the dynamics of spin-labelled BetP using pulsed electron-electron double resonance (PELDOR) under different stimuli. We then carried out molecular simulations of structures of the BetP monomer to interpret the PELDOR data, using the enhanced-sampling methodology EBMetaD (1), whereby the dynamics of the protein are minimally biased so as to reproduce the experimental data. Comparison of the magnitude of the biasing work required for different input structures permitted us to assign them to specific states of the transport cycle under each of the experimental conditions. In particular, this analysis showed that BetP adopts inward-facing conformations in the presence of excess sodium, and a mixture of states when betaine is added. These studies better delineate the major conformations adopted by BetP in its transport cycle, and therefore provide important insights into its mechanism. More broadly, we illustrate how integrative simulations can aid interpretation of ambiguous structural and spectroscopic data on membrane proteins.\n\nAbbreviations

biophysics↗

bioRxiv2018

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT.

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy in transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the Neurotransmitter:Sodium Symporter (NSS) family, Na+ stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na+ transport. In a step essential for coupled transport, substrate binding must overcome the effect of Na+, allowing intracellular substrate and Na+ release from an inward-open state. However, it is unclear which specific elements of the protein mediate this conformational response to substrate binding. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na+ on conformation occurs at the Na2 site, where it influences conformation by fostering interaction between two domains of the protein (JBC 291: 1456, 2016). Here, we identify a conserved tyrosine residue in the substrate binding site required for substrate to enable conversion to inward-open states by establishing an interaction between the two transporter domains. We further identify additional interactions between the two transporter domains in the extracellular pathway that are required. Together with our previous work on the conformational effect of Na+, these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.

biochemistry↗