Promiscuous Binding in a Selective Protein: The Bacterial Na+/H+ Antiporter

The ability to discriminate between highly similar substrates is one of the remarkable properties of enzymes. For example, transporters and channels that selectively distinguish between various solutes enable living organisms to maintain and control their internal environment in the face of a constantly changing surrounding. Herein, we examine in detail the selectivity properties of one of the most important salt transporters: the bacterial Na/H antiporter. Selectivity can be achieved at either the substrate binding step or in subsequent antiporting. Surprisingly, using both computational and experimental analyses synergistically, we show that binding per se is not a sufficient determinant of selectively. All alkali ions from Li to Cs were able to competitively bind the antiporter's binding site, whether the protein was capable of pumping them or not. Hence, we propose that NhaA's binding site is relatively promiscuous and that the selectivity is determined at a later stage of the transport cycle

Extracellular mutation induces an allosteric effect across the membrane and hampers the activity of MRP1 (ABCC1)

Abstract Dynamic conformational changes play a major role in the function of proteins, including the ATP-Binding Cassette (ABC) transporters. Multidrug Resistance Protein 1 (MRP1) is an ABC exporter that protects cells from toxic molecules. Overexpression of MRP1 has been shown to confer Multidrug Resistance (MDR), a phenomenon in which cancer cells are capable to defend themselves against a broad variety of drugs. In this study, we used varied computational techniques to explore the unique F583A mutation that is known to essentially lock the transporter in a low-affinity solute binding state. We demonstrate how macro-scale conformational changes affect MRP1’s stability and dynamics, and how these changes correspond to micro-scale structural perturbations in helices 10–11 and the nucleotide-binding domains (NBDs) of the protein in regions known to be crucial for its ATPase activity. We demonstrate how a single substitution of an outward-facing aromatic amino acid causes a long-range allosteric effect that propagates across the membrane, ranging from the extracellular ECL5 loop to the cytoplasmic NBD2 over a distance of nearly 75 Å, leaving the protein in a non-functional state, and provide the putative allosteric pathway. The identified allosteric structural pathway is not only in agreement with experimental data but enhances our mechanical understanding of MRP1, thereby facilitating the rational design of chemosensitizers toward the success of chemotherapy treatments

Characterization of the Na + /H + Antiporter from Yersinia pestis

Yersinia pestis, the bacterium that historically accounts for the Black Death epidemics, has nowadays gained new attention as a possible biological warfare agent. In this study, its Na z /H z antiporter is investigated for the first time, by a combination of experimental and computational methodologies. We determined the protein’s substrate specificity and pH dependence by fluorescence measurements in everted membrane vesicles. Subsequently, we constructed a model of the protein’s structure and validated the model using molecular dynamics simulations. Taken together, better understanding of the Yersinia pestis Na z /H z antiporter’s structure-function relationship may assist in studies on ion transport, mechanism of action and designing specific blockers of Na z /H z antiporter to help in fighting Yersinia pestis-associated infections. We hope that our model will prove useful both from mechanistic and pharmaceutical perspectives

Demonstrating aspects of multiscale modeling by studying the permeation pathway of the human ZnT2 zinc transporter.

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

Correction: Demonstrating aspects of multiscale modeling by studying the permeation pathway of the human ZnT2 zinc transporter.

[This corrects the article DOI: 10.1371/journal.pcbi.1006503.]

Acetylation of Lysine 382 and Phosphorylation of Serine 392 in p53 Modulate the Interaction between p53 and MDC1 <i>In Vitro</i>

<div><p>Occurrence of DNA damage in a cell activates the DNA damage response, a survival mechanism that ensures genomics stability. Two key members of the DNA damage response are the tumor suppressor p53, which is the most frequently mutated gene in cancers, and MDC1, which is a central adaptor that recruits many proteins to sites of DNA damage. Here we characterize the <i>in vitro</i> interaction between p53 and MDC1 and demonstrate that p53 and MDC1 directly interact. The p53-MDC1 interaction is mediated by the tandem BRCT domain of MDC1 and the C-terminal domain of p53. We further show that both acetylation of lysine 382 and phosphorylation of serine 392 in p53 enhance the interaction between p53 and MDC1. Additionally, we demonstrate that the p53-MDC1 interaction is augmented upon the induction of DNA damage in human cells. Our data suggests a new role for acetylation of lysine 382 and phosphorylation of serine 392 in p53 in the cellular stress response and offers the first evidence for an interaction involving MDC1 that is modulated by acetylation. </p> </div

A C-terminus region (a.a. 318-393) of p53 directly binds MDC1-tBRCT.

<p>(a) His-tBRCT retrieves p53 fragments consisting a.a. 318-393: Bacterially expressed His-tBRCT was incubated with different radio-labeled fragments of p53-HA expressed in reticulocytes (for details see schematic representations below). Following His pull-down reactions the labeled p53 fragments (in the input or those retrieved by His-tBRCT) were visualized by autoradiography. (b) p53 fragments containing a.a 318-393 bind tBRCT-MDC1: Fragments of p53 fused to GST (for details see schematic representations below) were expressed in bacteria and purified. Following incubation with radio-labeled His-tBRCT and GST pull-down reactions, His-tBRCT visualized by using autoradiography. Input is 5% of His-tBRCT added to the reaction. The same gels were used for autoradiography and Coomassie blue staining in B. (c) GST pull-down using a.a. 318-393 of p53 fused to GST (GST-p53Cter) for His-FHA or His-tBRCT, followed by Coomassie blue staining. </p

In p53 CTD, acetylation of K382, phosphorylation of S392 or both, contribute to the binding to MDC1-tBRCT.

<p>(a) Cartoon representations of the p53 CTD peptides (Acetylated and phosphorylated peptide in blue or unmodified peptide in red), MDC1-tBRCT in cyan and phosphorous atom in gold. Left - initial conformations; right - representative snapshots of the molecular dynamics simulations. (b-e) Potential energy of the interactions between p53 CTD peptides and MDC1-tBRCT; The Lennard-Jones and the electrostatic contributions of each residue are shown in white and black, respectively. The peptides: (b) Ac-K382 and pS392. (c) Ac-K382. (d) pS392. (e) Unmodified. Error bars represent the standard deviation of the mean for the sum of the interactions.</p

Suggested model and visualization of the p53-MDC1 interaction.

<p>(a) MD derived interactions. tBRCT is shown in surface representation in light gray and the p53 CTD peptide is shown as a blue ribbon. Zoom-in panels of the Ac-K382 and pS392 are shown below; note that in the zoom-in panels the viewer angle is slightly rotated for visualization convenience. Red arrow points K1936 in MDC1. (b) Following genotoxic stress p53 (blue) undergoes K382 acetylation (red pentagon) and S392 phosphorylation (yellow pentagon). These residues mediate the interaction with MDC1 (gray) through its tBRCT domain (light gray). </p