Cristina Fenollar-Ferrer

Peptide aptamers targeting mutant p53 induce apoptosis in tumor cells.

Mutations in the p53 tumor suppressor gene frequently result in expression of p53 point mutants that accumulate in cancer cells and actively collaborate with tumor progression through the acquisition of novel properties. Interfering with mutant p53 functions may represent a valid alternative for blocking tumor growth and development of aggressive phenotypes. The interactions and activities of selected proteins can be specifically modulated by the binding of peptide aptamers (PA). In the present work, we isolated PAs able to interact more efficiently with p53 conformational mutants compared with wild-type p53. The interaction between mutant p53 and PAs was further characterized using molecular modeling. Transient expression of PAs was able to reduce the transactivation activity of mutant p53 and to induce apoptosis specifically in cells expressing mutant p53. These PAs could provide a potential strategy to inhibit the oncogenic functions of mutant p53 and improve mutant p53-targeted cancer therapies.

Structure and regulatory interactions of the cytoplasmic terminal domains of serotonin transporter.

Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein–protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional 1H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the “rocking bundle” hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.

Structural Fold and Binding Sites of the Human Na+-Phosphate Cotransporter NaPi-II

Phosphate plays essential biological roles and its plasma level in humans requires tight control to avoid bone loss (insufficiency) or vascular calcification (excess). Intestinal absorption and renal reabsorption of phosphate are mediated by members of the SLC34 family of sodium-coupled transporters (NaPi-IIa,b,c) whose membrane expression is regulated by various hormones, circulating proteins, and phosphate itself. Consequently, NaPi-II proteins are also potentially important pharmaceutical targets for controlling phosphate levels. Their crucial role in Pi homeostasis is underscored by pathologies resulting from naturally occurring SLC34 mutations and SLC34 knockout animals. SLC34 isoforms have been extensively studied with respect to transport mechanism and structure-function relationships; however, the three-dimensional structure is unknown. All SLC34 transporters share a duplicated motif comprising a glutamine followed by a stretch of threonine or serine residues, suggesting the presence of structural repeats as found in other transporter families. Nevertheless, standard bioinformatic approaches fail to clearly identify a suitable template for molecular modeling. Here, we used hydrophobicity profiles and hidden Markov models to define a structural repeat common to all SLC34 isoforms. Similar approaches identify a relationship with the core regions in a crystal structure of Vibrio cholerae Na(+)-dicarboxylate transporter VcINDY, from which we generated a homology model of human NaPi-IIa. The aforementioned SLC34 motifs in each repeat localize to the center of the model, and were predicted to form Na(+) and Pi coordination sites. Functional relevance of key amino acids was confirmed by biochemical and electrophysiological analysis of expressed, mutated transporters. Moreover, the validity of the predicted architecture is corroborated by extensive published structure-function studies. The model provides key information for elucidating the transport mechanism and predicts candidate substrate binding sites.

Dissecting a regulatory calcium-binding site of CLC-K kidney chloride channels

The kidney and inner ear CLC-K chloride channels, which are involved in salt absorption and endolymph production, are regulated by extracellular Ca2+ in the millimolar concentration range. Recently, Gradogna et al. (2010. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201010455) identified a pair of acidic residues (E261 and D278) located in the loop between helices I and J as forming a putative intersubunit Ca2+-binding site in hClC-Ka. In this study, we sought to explore the properties of the binding site in more detail. First, we verified that the site is conserved in hClC-Kb and rClC-K1. In addition, we could confer Ca2+ sensitivity to the Torpedo marmorata ClC-0 channel by exchanging its I–J loop with that from ClC-Ka, demonstrating a direct role of the loop in Ca2+ binding. Based on a structure of a bacterial CLC and a new sequence alignment, we built homology models of ClC-Ka. The models suggested additional amino acids involved in Ca2+ binding. Testing mutants of these residues, we could restrict the range of plausible models and positively identify two more residues (E259 and E281) involved in Ca2+ coordination. To investigate cation specificity, we applied extracellular Zn2+, Mg2+, Ba2+, Sr2+, and Mn2+. Zn2+ blocks ClC-Ka as well as its Ca2+-insensitive mutant, suggesting that Zn2+ binds to a different site. Mg2+ does not activate CLC-Ks, but the channels are activated by Ba2+, Sr2+, and Mn2+ with a rank order of potency of Ca2+ > Ba2+ > Sr2+ = Mn2+ for the human CLC-Ks. Dose–response analysis indicates that the less potent Ba2+ has a lower affinity rather than a lower efficacy. Interestingly, rClC-K1 shows an altered rank order (Ca2+ > Sr2+ >> Ba2+), but homology models suggest that residues outside the I–J loop are responsible for this difference. Our detailed characterization of the regulatory Ca2+-binding site provides a solid basis for the understanding of the physiological modulation of CLC-K channel function in the kidney and inner ear.

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called “repeat-swap modeling” to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism.

Identification of the First Sodium Binding Site of the Phosphate Cotransporter NaPi-IIa (SLC34A1)

Transporters of the SLC34 family (NaPi-IIa,b,c) catalyze uptake of inorganic phosphate (Pi) in renal and intestinal epithelia. The transport cycle requires three Na(+) ions and one divalent Pi to bind before a conformational change enables translocation, intracellular release of the substrates, and reorientation of the empty carrier. The electrogenic interaction of the first Na(+) ion with NaPi-IIa/b at a postulated Na1 site is accompanied by charge displacement, and Na1 occupancy subsequently facilitates binding of a second Na(+) ion at Na2. The voltage dependence of cotransport and presteady-state charge displacements (in the absence of a complete transport cycle) are directly related to the molecular architecture of the Na1 site. The fact that Li(+) ions substitute for Na(+) at Na1, but not at the other sites (Na2 and Na3), provides an additional tool for investigating Na1 site-specific events. We recently proposed a three-dimensional model of human SLC34a1 (NaPi-IIa) including the binding sites Na2, Na3, and Pi based on the crystal structure of the dicarboxylate transporter VcINDY. Here, we propose nine residues in transmembrane helices (TM2, TM3, and TM5) that potentially contribute to Na1. To verify their roles experimentally, we made single alanine substitutions in the human NaPi-IIa isoform and investigated the kinetic properties of the mutants by voltage clamp and (32)P uptake. Substitutions at five positions in TM2 and one in TM5 resulted in relatively small changes in the substrate apparent affinities, yet at several of these positions, we observed significant hyperpolarizing shifts in the voltage dependence. Importantly, the ability of Li(+) ions to substitute for Na(+) ions was increased compared with the wild-type. Based on these findings, we adjusted the regions containing Na1 and Na3, resulting in a refined NaPi-IIa model in which five positions (T200, Q206, D209, N227, and S447) contribute directly to cation coordination at Na1.

Role of β‐lactam carboxyl group on binding of penicillins and cephalosporins to class C β‐lactamases

Molecular models for the Henry Michaelis complexes of Enterobacter cloacae, a class C β‐lactamase, with penicillin G and cephalotin have been constructed by using molecular mechanic calculations, based on the AMBER force field, to examine the molecular differentiation mechanisms between cephalosporins and penicillins in β‐lactamases. Ser318Ala and Thr316Ala mutations in both complexes and Asn346Ala and Thr316Ala/Asn346Ala double mutation in penicillin G complex have also been studied. Results confirm that Thr316, Ser318, and Asn346 play a crucial role in the substrate recognition, via their interactions with one of the oxygens of the antibiotic carboxyl group. Both mutation Ser318Ala and Thr316Ala strongly affect the correct binding of cephalotin to P99, the first mainly by precluding the discriminating salt bridge between carboxyl and serine OH groups, and the second one by the Ser318, Lys315, and Tyr150 spatial rearrangements. On the other hand, Ser318Ala mutation has little effect on penicillin G binding, but the Thr316Ala/Asn346Ala double mutation causes the departure of the antibiotic from the oxyanion hole. Molecular dynamic simulations allow us to interpret the experimental results of some class C and A β‐lactamases. Proteins 2003;51:442–452.

Family resemblances: A common fold for some dimeric ion-coupled secondary transporters

The structures of two bacterial antiporters that act as multidrug resistance efflux pumps, MtrF and YdaH, resemble each other and that of the sodium-coupled succinate symporter VcINDY.

Cation Interactions and Membrane Potential Induce Conformational Changes in NaPi-IIb

Voltage-dependence of Na(+)-coupled phosphate cotransporters of the SLC34 family arises from displacement of charges intrinsic to the protein and the binding/release of one Na(+) ion in response to changes in the transmembrane electric field. Candidate coordination residues for the cation at the Na1 site were previously predicted by structural modeling using the x-ray structure of dicarboxylate transporter VcINDY as template and confirmed by functional studies. Mutations at Na1 resulted in altered steady-state and presteady-state characteristics that should be mirrored in the conformational changes induced by membrane potential changes. To test this hypothesis by functional analysis, double mutants of the flounder SLC34A2 protein were constructed that contain one of the Na1-site perturbing mutations together with a substituted cysteine for fluorophore labeling, as expressed in Xenopus oocytes. The locations of the mutations were mapped onto a homology model of the flounder protein. The effects of the mutagenesis were characterized by steady-state, presteady-state, and fluorometric assays. Changes in fluorescence intensity (ΔF) in response to membrane potential steps were resolved at three previously identified positions. These fluorescence data corroborated the altered presteady-state kinetics upon perturbation of Na1, and furthermore indicated concomitant changes in the microenvironment of the respective fluorophores, as evidenced by changes in the voltage dependence and time course of ΔF. Moreover, iodide quenching experiments indicated that the aqueous nature of the fluorophore microenvironment depended on the membrane potential. These findings provide compelling evidence that membrane potential and cation interactions induce significant large-scale structural rearrangements of the protein.

Insights on the acetylated NF-κB transcription factor complex with DNA from molecular dynamics simulations

The nuclear factor‐κB (NF‐κB) is a DNA sequence‐specific regulator of many important biological processes, whose activity is modulated by enzymatic acetylation. In one of the best functionally characterized NF‐κB complexes, the p50/p65 heterodimer, acetylation of K221 at p65 causes a decrease of DNA dissociation rate, whilst the acetylation of K122 and K123, also at p65, markedly decreases the binding affinity for DNA. By means of molecular dynamics simulations based on the X‐ray structure of the p50/p65 complex with DNA, we provide insights on the structural determinants of the acetylated complexes in aqueous solution. Lysine acetylation involves the loss of favorable electrostatic interactions between DNA and NF‐κB, which is partially compensated by the reduction of the desolvation free‐energy of the two binding partners. Acetylation at both positions K122 and K123 is associated with a decrease of the electrostatic potential at the p65/DNA interface, which is only partially counterbalanced by an increase of the local Na+ concentration. It induces the disruption of base‐specific and nonspecific interactions between DNA and NF‐κB and it is consistent with the observed decrease of binding affinity. In contrast, acetylation at position K221 results in the loss of nonspecific protein–DNA interactions, but the DNA recognition sites are not affected. In addition, the loss of protein–DNA interactions is likely to be counterbalanced by an increase of the configurational entropy of the complex, which provides, at a speculative level, a justification for the observed decrease of NF‐κB/DNA dissociation rate. Proteins 2012.