Blanca López-Méndez

Structural basis for the aminoacid composition of proteins from halophilic archea

In order to survive in highly saline environments, proteins from halophilic archea have evolved with biased amino acid compositions that have the capacity to reduce contacts with the solvent.

Protein Stabilization and the Hofmeister Effect: The Role of Hydrophobic Solvation

Using the IGg binding domain of protein L from Streptoccocal magnus (ProtL) as a case study, we investigated how the anions of the Hofmeister series affect protein stability. To that end, a suite of lysine-to-glutamine modifications were obtained and structurally and thermodynamically characterized. The changes in stability introduced with the mutation are related to the solvent-accessible area of the side chain, specifically to the solvation of the nonpolar moiety of the residue. The thermostability for the set of ProtL mutants was determined in the presence of varying concentrations (0-1 M) of six sodium salts from the Hofmeister series: sulfate, phosphate, fluoride, nitrate, perchlorate, and thiocyanate. For kosmotropic anions (sulfate, phosphate, and fluoride), the stability changes induced by the cosolute (encoded in m(3)=deltaDeltaG(0)/deltaC(3)) are proportional to the surface changes introduced with the mutation. In contrast, the m(3) values measured for chaotropic anions are much more independent of such surface modifications. Our results are consistent with a model in which the increase in the solution surface tension induced by the anion stabilizes the folded conformation of the protein. This contribution complements the nonspecific and weak interactions between the ions and the protein backbone that shift the equilibrium toward the unfolded state.

A Conserved Motif Provides Binding Specificity to the PP2A-B56 Phosphatase

Dynamic protein phosphorylation is a fundamental mechanism regulating biological processes in all organisms. Protein phosphatase 2A (PP2A) is the main source of phosphatase activity in the cell, but the molecular details of substrate recognition are unknown. Here, we report that a conserved surface-exposed pocket on PP2A regulatory B56 subunits binds to a consensus sequence on interacting proteins, which we term the LxxIxE motif. The composition of the motif modulates the affinity for B56, which in turn determines the phosphorylation status of associated substrates. Phosphorylation of amino acid residues within the motif increases B56 binding, allowing integration of kinase and phosphatase activity. We identify conserved LxxIxE motifs in essential proteins throughout the eukaryotic domain of life and in human viruses, suggesting that the motifs are required for basic cellular function. Our study provides a molecular description of PP2A binding specificity with broad implications for understanding signaling in eukaryotes.

Discovery of Selective Ligands for Telomeric RNA G-quadruplexes (TERRA) through 19F-NMR Based Fragment Screening

Telomeric repeat-containing RNA (TERRA) is a novel and very attractive antitumoral target. Here, we report the first successful application of (19)F-NMR fragment-based screening to identify chemically diverse compounds that bind to an RNA molecule such as TERRA. We have built a library of 355 fluorinated fragments, and checked their interaction with a long telomeric RNA as a target molecule. The screening resulted in the identification of 20 hits (hit rate of 5.6%). For a number of binders, their interaction with TERRA was confirmed by (19)F- and (1)H NMR as well as by CD melting experiments. We have also explored the selectivity of the ligands for RNA G-quadruplexes and found that some of the hits do not interact with other nucleic acids such as tRNA and duplex DNA and, most importantly, favor the propeller-like parallel conformation in telomeric DNA G-quadruplexes. This suggests a selective recognition of this particular quadruplex topology and that different ligands may recognize specific sites in propeller-like parallel G-quadruplexes. Such features make some of the resulting binders promising lead compounds for fragment based drug discovery.

BuD, a helix–loop–helix DNA-binding domain for genome modification

Crystal structures of BurrH and the BurrH–DNA complex are reported.

Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling

Proper segregation of chromosomes depends on a functional spindle assembly checkpoint (SAC) and requires kinetochore localization of the Bub1 and Mad1/Mad2 checkpoint proteins. Several aspects of Mad1/Mad2 kinetochore recruitment in human cells are unclear and in particular the underlying direct interactions. Here we show that conserved domain 1 (CD1) in human Bub1 binds directly to Mad1 and a phosphorylation site exists in CD1 that stimulates Mad1 binding and SAC signalling. Importantly, fusion of minimal kinetochore-targeting Bub1 fragments to Mad1 bypasses the need for CD1, revealing that the main function of Bub1 is to position Mad1 close to KNL1 MELT repeats. Furthermore, we identify residues in Mad1 that are critical for Mad1 functionality, but not Bub1 binding, arguing for a direct role of Mad1 in the checkpoint. This work dissects functionally relevant molecular interactions required for spindle assembly checkpoint signalling at kinetochores in human cells.

Conformational Selection and Induced Fit Mechanisms in the Binding of an Anticancer Drug to the c-Src Kinase.

Understanding the conformational changes associated with the binding of small ligands to their biological targets is a fascinating and meaningful question in chemistry, biology and drug discovery. One of the most studied and important is the so-called “DFG-flip” of tyrosine kinases. The conserved three amino-acid DFG motif undergoes an “in to out” movement resulting in a particular inactive conformation to which “type II” kinase inhibitors, such as the anti-cancer drug Imatinib, bind. Despite many studies, the details of this prototypical conformational change are still debated. Here we combine various NMR experiments and surface plasmon resonance with enhanced sampling molecular dynamics simulations to shed light into the conformational dynamics associated with the binding of Imatinib to the proto-oncogene c-Src. We find that both conformational selection and induced fit play a role in the binding mechanism, reconciling opposing views held in the literature. Moreover, an external binding pose and local unfolding (cracking) of the aG helix are observed.

A PTIP–PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex

Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.

De Novo Designed Library of Linear Helical Peptides: An Exploratory Tool in the Discovery of Protein–Protein Interaction Modulators

Protein-protein interactions (PPIs) have emerged as important targets for pharmaceutical intervention because of their essential role in numerous physiological and pathological processes, but screening efforts using small-molecules have led to very low hit rates. Linear peptides could represent a quick and effective approach to discover initial PPI hits, particularly if they have inherent ability to adopt specific peptide secondary structures. Here, we address this hypothesis through a linear helical peptide library, composed of four sublibraries, which was designed by theoretical predictions of helicity (Agadir software). The 13-mer peptides of this collection fixes either a combination of three aromatic or two aromatic and one aliphatic residues on one face of the helix (Ac-SSEEX(5)ARNX(9)AAX(12)N-NH2), since these are structural features quite common at PPIs interfaces. The 81 designed peptides were conveniently synthesized by parallel solid-phase methodologies, and the tendency of some representative library components to adopt the intended secondary structure was corroborated through CD and NMR experiments. As proof of concept in the search for PPI modulators, the usefulness of this library was verified on the widely studied p53-MDM2 interaction and on the communication between VEGF and its receptor Flt-1, two PPIs for which a hydrophobic α-helix is essential for the interaction. We have demonstrated here that, in both cases, selected peptides from the library, containing the right hydrophobic sequence of the hot-spot in one of the protein partners, are able to interact with the complementary protein. Moreover, we have discover some new, quite potent inhibitors of the VEGF-Flt-1 interaction, just by replacing one of the aromatic residues of the initial F(5)Y(9)Y(12) peptide by W, in agreement with previous results on related antiangiogenic peptides. Finally, the HTS evaluation of the full collection on thermoTRPs has led to a few antagonists of TRPV1 and TRPA1 channels, which open new avenues on the way to innovative modulators of these channels.

Allosteric regulation of Csx1, a type IIIB-associated CARF domain ribonuclease by RNAs carrying a tetraadenylate tail

Abstract CRISPR–Cas systems protect prokaryotes against invading viruses and plasmids. The system is associated with a large number of Cas accessory proteins among which many contain a CARF (CRISPR-associated Rossmann fold) domain implicated in ligand binding and a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) nuclease domain. Here, such a dual domain protein, i.e. the Sulfolobus islandicus Csx1 (SisCsx1) was characterized. The enzyme exhibited metal-independent single-strand specific ribonuclease activity. In fact, SisCsx1 showed a basal RNase activity in the absence of ligand; upon the binding of an RNA ligand carrying four continuous adenosines at the 3′-end (3′-tetra-rA), the activated SisCsx1 degraded RNA substrate with a much higher turnover rate. Amino acid substitution mutants of SisCsx1 were obtained, and characterization of these mutant proteins showed that the CARF domain of the enzyme is responsible for binding to 3′-tetra-rA and the ligand binding strongly activates RNA cleavage by the HEPN domain. Since RNA polyadenylation is an important step in RNA decay in prokaryotes, and poly(A) RNAs can activate CARF domain proteins, the poly(A) RNA may function as an important signal in the cellular responses to viral infection and environmental stimuli, leading to degradation of both viral and host transcripts and eventually to cell dormancy or cell death.