Biology subjects

Flores-Canales, J. C.

Publications and source records attributed to Flores-Canales, J. C..

3 recordsLinked to original sources

bioRxiv2020

Molecular mechanism of high affinity sugar transport in plants unveiled by structures of glucose/H+ symporter STP10

Sugars are essential sources of energy and carbon, and also function as key signaling molecules in plants. Sugar Transport Proteins (STP) are proton-coupled symporters, solely responsible for uptake of glucose from the apoplastic compartment into cells in all plant tissues. They are integral to organ development in symplastically isolated tissues such as seeds, pollen and fruit. Additionally, STPs play a significant role in plant responses to both environmental stressors such as dehydration, and prevalent fungal infections like rust and mildew. Here, we present two high-resolution crystal structures of the outward-occluded and inward-open conformations of Arabidopsis thaliana STP10 with glucose and protons bound. The two structures describe key states in the STP transport cycle. Together with in vivo biochemical analysis and Molecular Dynamics simulations they pinpoint structural elements that explain how STPs exhibit high affinity for sugar binding on the extracellular side and how it is considerably lowered on the intracellular side to facilitate substrate release. These structural elements, conserved in all STPs across plant species, clarify the basis of proton-to-glucose coupling, essential for symport. The results advance our understanding of a key molecular mechanism behind plant organ development, and sets the stage for novel bioengineering strategies in crops that could target seeds, fruits and plant resistance to fungal infections.

plant biology↗

bioRxiv2020

Substrate Recognition by γ-Glutamyltransferase: A Molecular Dynamics Simulations Study

{gamma}-glutamyltransferase (GGT) is an enzyme that uses {gamma}-glutamyl compounds as substrate and catalyzes their transfer into a water molecule or an acceptor substrate with varied physiological-function in bacteria, plants and animals. Crystal structures of GGT are known for different species and in different states of the chemical reaction; however, structural dynamics of the substrate binding to the catalytic site of GGT is unknown. Here, we modeled Escherichia Coli GGTs glutamine binding by using a swarm of accelerated molecular dynamics (aMD) simulations. Characterization of multiple binding events identified three structural binding motifs composed of polar residues in the binding pocket that govern glutamine binding into the active site. Simulated open and closed conformations of a lid-loop protecting the binding cavity suggests its role as a gating element by allowing or blocking substrates entry into the binding pocket. Partially open states of the lid-loop are accessible within thermal fluctuations, while the estimated free energy cost of a complete open state is 2.4 kcal/mol. Our results suggest that both specific electrostatic interactions and GGT conformational dynamics dictate the molecular recognition of substrate-GGT complexes.

biophysics↗

bioRxiv2019

Microsecond Molecular Dynamics Simulations of Diphtheria Toxin Translocation T-Domain pH-Dependent Unfolding in Solution

Diphtheria toxin is a multi-domain protein that invades cells by using their own endocytosis mechanism. In endocytosis, an endosome, a lipid bilayer vesicle, is formed to encapsulate an extracellular molecule. Subsequent acidification of endosome internal solution induces conformational rearrangements and membrane insertion of such encapsulated diphtheria toxin translocation domain (T-domain). In solution at neutral pH, a stand-alone T-domain adopts an all alpha-helical globular structure; however, atomistic details of the pH-dependent conformational changes of the protein are not completely understood. We model structural rearrangements in T-domain in 18 {micro}s long molecular dynamics (MD) simulations of neutral and low pH T-domain models in explicit solvent. At low pH, six histidine residues of the protein were protonated. Two independent MD trajectories resulted in partial protein unfolding at low pH, in which similar regions of the protein conformational subspace were explored. Notably, a pH induced unfolding transition was initiated by partial unfolding of helix TH4 followed by unfolding of helix TH1. Helix TH2 repeatedly unfolds in the low pH T-domain model, which is consequently predicted to be disordered by a consensus of disorder prediction algorithms. Protonation of histidines disrupted a hydrophobic core containing a putative transmembrane helix TH8, which is encircled by hydrophobic surfaces of helices TH3, TH5 and TH9. Afterwards, the low pH T-domain model was reorganized into an ensemble of partially unfolded structures with increased solvent exposure of hydrophobic and charged sites. Thus, MD simulations suggest the destabilizing role of protonation of histidines, in the neutral pH conformation in solution, which may facilitate the initial stages of T-domain membrane binding. The simulation at neutral pH samples conformations in the vicinity of the native structure of the protein. However, significant fluctuations of the protein, including unfolding and refolding of -helices were observed at these simulation time-scales.

biophysics↗