David Pantoja-Uceda

A Conserved Docking Surface on Calcineurin Mediates Interaction with Substrates and Immunosuppressants

The phosphatase calcineurin, a target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIxIT and LxVP sites. Although many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and we demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATC1 and the interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates, and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.

Molecular Basis of Histone H3K4me3 Recognition by ING4

The inhibitors of growth (ING) family of tumor suppressors consists of five homologous proteins involved in chromatin remodeling. They form part of different acetylation and deacetylation complexes and are thought to direct them to specific regions of the chromatin, through the recognition of H3K4me3 (trimethylated K4 in the histone 3 tail) by their conserved plant homeodomain (PHD). We have determined the crystal structure of ING4-PHD bound to H3K4me3, which reveals a tight complex stabilized by numerous interactions. NMR shows that there is a reduction in the backbone mobility on the regions of the PHD that participate in the peptide binding, and binding affinities differ depending on histone tail lengths Thermodynamic analysis reveals that the discrimination in favor of methylated lysine is entropy-driven, contrary to what has been described for chromodomains. The molecular basis of H3K4me3 recognition by ING4 differs from that of ING2, which is consistent with their different affinities for methylated histone tails. These differences suggest a distinct role in transcriptional regulation for these two ING family members because of the antagonistic effect of the complexes that they recruit onto chromatin. Our results illustrate the versatility of PHD fingers as readers of the histone code.

The TDP-43 N-terminal domain structure at high resolution.

Transactive response DNA‐binding protein 43 kDa (TDP‐43) is an RNA transporting and processing protein whose aberrant aggregates are implicated in neurodegenerative diseases. The C‐terminal domain of this protein plays a key role in mediating this process. However, the N‐terminal domain (residues 1–77) is needed to effectively recruit TDP‐43 monomers into this aggregate. In the present study, we report, for the first time, the essentially complete 1H, 15N and 13C NMR assignments and the structure of the N‐terminal domain determined on the basis of 26 hydrogen‐bond, 60 torsion angle and 1058 unambiguous NOE structural restraints. The structure consists of an α‐helix and six β‐strands. Two β‐strands form a β‐hairpin not seen in the ubiquitin fold. All Pro residues are in the trans conformer and the two Cys are reduced and distantly separated on the surface of the protein. The domain has a well defined hydrophobic core composed of F35, Y43, W68, Y73 and 17 aliphatic side chains. The fold is topologically similar to the reported structure of axin 1. The protein is stable and no denatured species are observed at pH 4 and 25 °C. At 4 kcal·mol−1, the conformational stability of the domain, as measured by hydrogen/deuterium exchange, is comparable to ubiquitin (6 kcal·mol−1). The β‐strands, α‐helix, and three of four turns are generally rigid, although the loop formed by residues 47–53 is mobile, as determined by model‐free analysis of the 15N{1H}NOE, as well as the translational and transversal relaxation rates.

Amino acid type identification in NMR spectra of proteins via β- and γ-carbon edited experiments

In this work, we introduce a set of pulse sequences that provide amino acid type identification of the NH correlation signals of proteins. The first pulse sequence is a modification of the CBCA(CO)NH experiment that exploits spin-coupling topologies to differentiate between amino acid types. A set of eight 2D (1)H-(15)N correlation spectra is recorded where the sign of the cross-peaks change from one spectrum to another according to the amino acid type of the preceding residue in the protein sequence. Linear combination of these eight data sets produces four subspectra. Taking also into account the sign of the correlation signals, this method allows the classification of the NH signals into six different groups, depending on the character of the preceding residue. This sequence is complemented with a (CGCBCACO)NH experiment that allows the subdivision of the largest of these groups into two smaller ones. Finally, a modification of the CBCANH experiment led to a similar classification of NH signals into six different groups, but now depending on the type of its own amino acid. The set of pulse sequences is demonstrated with two proteins of small to moderate size.

De novo Design of Monomeric β-Hairpin and β-Sheet Peptides

: Since the first report in 1993 (JACS 115, 5887-5888) of a peptide able to form a monomeric beta-hairpin structure in aqueous solution, the design of peptides forming either beta-hairpins (two-stranded antiparallel beta-sheets) or three-stranded antiparallel beta-sheets has become a field of intense interest. These studies have yielded great insights into the principles governing the stability and folding of beta-hairpins and antiparallel beta-sheets. This chapter reviews briefly those principles and describes a protocol for the de novo design of beta-sheet-forming peptides based on them. Criteria to select appropriate turn and strand residues and to avoid aggregation are provided. Because nuclear magnetic resonance is the most appropriate technique to check the success of new designs, the nuclear magnetic resonance parameters characteristic of beta-hairpins and three-stranded antiparallel beta-sheets are given.

Deciphering the structural basis that guides the oxidative folding of leech-derived tryptase inhibitor.

Protein folding mechanisms have remained elusive mainly because of the transient nature of intermediates. Leech-derived tryptase inhibitor (LDTI) is a Kazal-type serine proteinase inhibitor that is emerging as an attractive model for folding studies. It comprises 46 amino acid residues with three disulfide bonds, with one located inside a small triple-stranded antiparallel β-sheet and with two involved in a cystine-stabilized α-helix, a motif that is widely distributed in bioactive peptides. Here, we analyzed the oxidative folding and reductive unfolding of LDTI by chromatographic and disulfide analyses of acid-trapped intermediates. It folds and unfolds, respectively, via sequential oxidation and reduction of the cysteine residues that give rise to a few 1- and 2-disulfide intermediates. Species containing two native disulfide bonds predominate during LDTI folding (IIa and IIc) and unfolding (IIa and IIb). Stop/go folding experiments demonstrate that only intermediate IIa is productive and oxidizes directly into the native form. The NMR structures of acid-trapped and further isolated IIa, IIb, and IIc reveal global folds similar to that of the native protein, including a native-like canonical inhibitory loop. Enzyme kinetics shows that both IIa and IIc are inhibitory-active, which may substantially reduce proteolysis of LDTI during its folding process. The results reported show that the kinetics of the folding reaction is modulated by the specific structural properties of the intermediates and together provide insights into the interdependence of conformational folding and the assembly of native disulfides during oxidative folding.

Direct correlation of consecutive C′–N groups in proteins: a method for the assignment of intrinsically disordered proteins

Two novel 3D 13C-detected experiments, hNcocaNCO and hnCOcaNCO, are proposed to facilitate the resonance assignment of intrinsically disordered proteins. The experiments correlate the 15N and 13C′ chemical shifts of two consecutive amide moieties without involving other nuclei, thus taking advantage of the good dispersion shown by the 15N–13C′ correlations, even for proteins that lack a well defined tertiary structure. The new pulse sequences were successfully tested using Nupr1, an intrinsically disordered protein of 93 residues.

Solution structure of the rhodanese homology domain At4g01050(175–295) from Arabidopsis thaliana

The three‐dimensional structure of the rhodanese homology domain At4g01050(175–195) from Arabidopsis thaliana has been determined by solution nuclear magnetic resonance methods based on 3043 upper distance limits derived from NOE intensities measured in three‐dimensional NOESY spectra. The structure shows a backbone root mean square deviation to the mean coordinates of 0.43 Å for the structured residues 7–125. The fold consists of a central parallel β‐sheet with five strands in the order 1–5–4–2–3 and arranged in the conventional counterclockwise twist, and helices packing against each side of the β‐sheet. Comparison with the sequences of other proteins with a rhodanese homology domain in Arabidopsis thaliana indicated residues that could play an important role in the scaffold of the rhodanese homology domain. Finally, a three‐dimensional structure comparison of the present noncatalytic rhodanese homology domain with the noncatalytic rhodanese domains of sulfurtransferases from other organisms discloses differences in the length and conformation of loops that could throw light on the role of the noncatalytic rhodanese domain in sulfurtransferases.

Proliferating cell nuclear antigen (PCNA) interactions in solution studied by NMR.

PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86 kDa by the symmetric association of three identical protomers. The ring encircles the DNA and acts as a docking platform for other proteins, most of them containing the PCNA Interaction Protein sequence (PIP-box). We have used NMR to characterize the interactions of PCNA with several other proteins and fragments in solution. The binding of the PIP-box peptide of the cell cycle inhibitor p21 to PCNA is consistent with the crystal structure of the complex. A shorter p21 peptide binds with reduced affinity but retains most of the molecular recognition determinants. However the binding of the corresponding peptide of the tumor suppressor ING1 is extremely weak, indicating that slight deviations from the consensus PIP-box sequence dramatically reduce the affinity for PCNA, in contrast with a proposed less stringent PIP-box sequence requirement. We could not detect any binding between PCNA and the MCL-1 or the CDK2 protein, reported to interact with PCNA in biochemical assays. This suggests that they do not bind directly to PCNA, or they do but very weakly, with additional unidentified factors stabilizing the interactions in the cell. Backbone dynamics measurements show three PCNA regions with high relative flexibility, including the interdomain connector loop (IDCL) and the C-terminus, both of them involved in the interaction with the PIP-box. Our work provides the basis for high resolution studies of direct ligand binding to PCNA in solution.

Solution structure of human growth arrest and DNA damage 45alpha (Gadd45alpha) and its interactions with proliferating cell nuclear antigen (PCNA) and Aurora A kinase

Gadd45α is a nuclear protein encoded by a DNA damage-inducible gene. Through its interactions with other proteins, Gadd45α participates in the regulation of DNA repair, cell cycle, cell proliferation, and apoptosis. The NMR structure of human Gadd45α has been determined and shows an α/β fold with two long disordered and flexible regions at the N terminus and one of the loops. Human Gadd45α is predominantly monomeric in solution but exists in equilibrium with dimers and other oligomers whose population increases with protein concentration. NMR analysis shows that Aurora A interacts through its N-terminal domain with a region of human Gadd45α encompassing the site of dimerization, suggesting that the oligomerization of Gadd45α could be a regulatory mechanism to modulate its interactions with Aurora A, and possibly with other proteins too. However, Gadd45α appears to interact only weakly with PCNA through its flexible loop, in contrast with previous and contradictory reports.