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Polyglutamine (polyQ) tracts are regions of low sequence complexity frequently found in transcription factors. Tract length often correlates with transcriptional activity and expansion beyond specific thresholds in certain human proteins is the cause of polyQ disorders. To study the structural basis of the association between tract length, transcriptional activity and disease, we addressed how the conformation of the polyQ tract of the androgen receptor, associated with spinobulbar muscular atrophy (SBMA), depends on its length. Here we report that this sequence folds into a helical structure stabilized by unconventional hydrogen bonds between glutamine side chains and main chain carbonyl groups, and that its helicity directly correlates with tract length. These unusual hydrogen bonds are bifurcate with the conventional hydrogen bonds stabilizing α-helices. Our findings suggest a plausible rationale for the association between polyQ tract length and androgen receptor transcriptional activity and have implications for establishing the mechanistic basis of SBMA. Polyglutamine (polyQ) tracts are low-complexity regions and their expansion is linked to certain neurodegenerative diseases. Here the authors combine experimental and computational approaches to find that the length of the androgen receptor polyQ tract correlates with its helicity and show that the polyQ helical structure is stabilized by hydrogen bonds between the Gln side chains and main chain carbonyl groups.

Prototypic receptors for human influenza viruses are N -glycans carrying α2,6-linked sialosides. Due to immune pressure, A/H3N2 influenza viruses have emerged with altered receptor specificities that bind α2,6-linked sialosides presented on extended N -acetyl-lactosamine (LacNAc) chains. Here, binding modes of such drifted hemagglutinin’s (HAs) are examined by chemoenzymatic synthesis of N -glycans having 13 C-labeled monosaccharides at strategic positions. The labeled glycans are employed in 2D STD- 1 H by 13 C-HSQC NMR experiments to pinpoint which monosaccharides of the extended LacNAc chain engage with evolutionarily distinct HAs. The NMR data in combination with computation and mutagenesis demonstrate that mutations distal to the receptor binding domain of recent HAs create an extended binding site that accommodates with the extended LacNAc chain. A fluorine containing sialoside is used as NMR probe to derive relative binding affinities and confirms the contribution of the extended LacNAc chain for binding. Binding modes of antigenically drifted hemagglutinins of human influenza A viruses have been determined by NMR using synthetic N-glycans having 13 C-labeled monosaccharides to pinpoint which monosaccharides of extended LacNAc chains engage with the HAs.

The binding of intrinsically disordered proteins to globular ones can require the folding of motifs into α-helices. These interactions offer opportunities for therapeutic intervention but their modulation with small molecules is challenging because they bury large surfaces. Linear peptides that display the residues that are key for binding can be targeted to globular proteins when they form stable helices, which in most cases requires their chemical modification. Here we present rules to design peptides that fold into single α-helices by instead concatenating glutamine side chain to main chain hydrogen bonds recently discovered in polyglutamine helices. The resulting peptides are uncharged, contain only natural amino acids, and their sequences can be optimized to interact with specific targets. Our results provide design rules to obtain single α-helices for a wide range of applications in protein engineering and drug design. Targeting biomedically relevant protein-protein interactions is a long-lasting challenge in medicinal chemistry. Here, the authors develop a single α-helical peptide scaffold that can be tailored to target globular proteins of biomedical interest.

Background Metabolic syndrome (MetS) is a multimorbid long-term condition without consensual medical definition and a diagnostic based on compatible symptomatology. Here we have investigated the molecular signature of MetS in urine. Methods We used NMR-based metabolomics to investigate a European cohort including urine samples from 11,754 individuals (18–75 years old, 41% females), designed to populate all the intermediate conditions in MetS, from subjects without any risk factor up to individuals with developed MetS (4–5%, depending on the definition). A set of quantified metabolites were integrated from the urine spectra to obtain metabolic models (one for each definition), to discriminate between individuals with MetS. Results MetS progression produces a continuous and monotonic variation of the urine metabolome, characterized by up- or down-regulation of the pertinent metabolites (17 in total, including glucose, lipids, aromatic amino acids, salicyluric acid, maltitol, trimethylamine N -oxide, and p -cresol sulfate) with some of the metabolites associated to MetS for the first time. This metabolic signature, based solely on information extracted from the urine spectrum, adds a molecular dimension to MetS definition and it was used to generate models that can identify subjects with MetS (AUROC values between 0.83 and 0.87). This signature is particularly suitable to add meaning to the conditions that are in the interface between healthy subjects and MetS patients. Aging and non-alcoholic fatty liver disease are also risk factors that may enhance MetS probability, but they do not directly interfere with the metabolic discrimination of the syndrome. Conclusions Urine metabolomics, studied by NMR spectroscopy, unravelled a set of metabolites that concomitantly evolve with MetS progression, that were used to derive and validate a molecular definition of MetS and to discriminate the conditions that are in the interface between healthy individuals and the metabolic syndrome.

The intrinsically disordered protein p15 PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15 PAF –PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15 PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15 PAF also contacts the inside of, and passes through, the PCNA ring. The disordered p15 PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15 PAF binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15 PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding. p15PAF regulates DNA replication and repair via interactions with the Proliferating Cell Nuclear Antigen (PCNA) sliding clamp. Here the authors present multi-faceted structural analyses of the p15-PCNA-DNA complex that suggests p15 regulates the sliding of PCNA along DNA during replication.

The accepted model for the interaction of α and β integrins in the transmembrane (TM) domain is based on the pair αIIbβ3. This involves the so-called outer and inner membrane association clasps (OMC and IMC, respectively). In the α chain, the OMC involves a GxxxG-like motif, whereas in the IMC a conserved juxtamembrane GFFKR motif experiences a backbone reversal that partially fills the void generated by TM separation towards the cytoplasmic half. However, the GFFKR motif of several α integrin cytoplasmic tails in non-bicelle environments has been shown to adopt an α-helical structure that is not membrane-embedded and which was shown to bind a variety of cytoplasmic proteins. Thus it is not known if a membrane-embedded backbone reversal is a conserved structural feature in α integrins. We have studied the system αLβ2 because of its importance in leukocytes, where integrin deactivation is particularly important. Herein we show that the backbone reversal feature is not only present in αIIb but also in αL-TM when reconstituted in bicelles. Additionally, titration with β2 TM showed eight residues clustering along one side of αL-TM, forming a plausible interacting face with β2. The latter orientation is consistent with a previously predicted reported polar interaction between αL Ser-1071 and β2 Thr-686.

Fluorinated glycomimetics are frequently employed to study and eventually modulate protein–glycan interactions. However, complex glycans and their glycomimetics may display multiple binding epitopes that enormously complicate the access to a complete picture of the protein–ligand complexes. We herein present a new methodology based on the synergic combination of experimental 19F-based saturation transfer difference (STD) NMR data with computational protocols, applied to analyze the interaction between DC-SIGN, a key lectin involved in inflammation and infection events with the trifluorinated glycomimetic of the trimannoside core, ubiquitous in human glycoproteins. A novel 2D-STD-TOCSYreF NMR experiment was employed to obtain the experimental STD NMR intensities, while the Complete Relaxation Matrix Analysis (CORCEMA-ST) was used to predict that expected for an ensemble of geometries extracted from extensive MD simulations. Then, an in-house built computer program was devised to find the ensemble of structures that provide the best fit between the theoretical and the observed STD data. Remarkably, the experimental STD profiles obtained for the ligand/DC-SIGN complex could not be satisfactorily explained by a single binding mode, but rather with a combination of different modes coexisting in solution. Therefore, the method provides a precise view of those ligand–receptor complexes present in solution.

The metastasis suppressor KISS1 is reported to be involved in the progression of several solid neoplasias, making it a promising molecular target for controlling their metastasis. The KISS1 sequence contains an N-terminal secretion signal and several dibasic sequences that are proposed to be the proteolytic cleavage sites. We present the first structural characterization of KISS1 by circular dichroism, multi-angle light scattering, small angle X-Ray scattering and NMR spectroscopy. An analysis of the KISS1 backbone NMR chemical shifts does not reveal any preferential conformation and deviation from a random coil ensemble. The backbone 15N transverse relaxation times indicate a mildly reduced mobility for two regions that are rich in bulky residues. The small angle X-ray scattering curve of KISS1 is likewise consistent with a predominantly random coil ensemble, although an ensemble optimization analysis indicates some preference for more extended conformations possibly due to positive charge repulsion between the abundant basic residues. Our results support the hypothesis that KISS1 mostly samples a random coil conformational space, which is consistent with its high susceptibility to proteolysis and the generation of Kisspeptin fragments.

Ribosome biogenesis is an energetically expensive and complex cellular process that involves the coordinated folding of the ribosomal RNA and dozens of ribosomal proteins. It proceeds along multiple parallel pathways and is guided by trans-acting factors called ribosome assembly factors. Although this process has been studied for decades, there are still many open questions regarding the role of the ribosome assembly factors in directing the folding of ribosome biogenesis intermediates. RimP is one of the early acting factors and guides the assembly of the small 30S ribosomal subunit by facilitating the binding of ribosomal proteins uS5 and uS12. Here we report the virtually complete 1H, 15N, and 13C chemical shift assignment of RimP from Escherichia coli. The NMR chemical shift data, deposited in the BMRB data bank under Accession No. 28014, indicates a widely folded protein composed of three alpha helices and eight beta strands.

RbfA (ribosome binding factor A; 15.2 kDa) is a protein involved in ribosome biogenesis and has been shown to be important for growth at low temperatures and to act as a suppressor for a cold-sensitive mutation (C23U) in the ribosomal RNA of the small 30S ribosomal subunit. The 3D structure of isolated RbfA has been determined from several organisms showing that RbfA has type-II KH-domain fold topology similar to the KH domain of another assembly factor, Era, whose overexpression can compensate for the deletion of rbfA, suppressing both the cold sensitivity and abnormal accumulation of 17S rRNA in rbfA knockout stains. Interestingly, a RbfAΔ25 variant used in previous NMR studies, truncated at the C-terminal domain to remove 25 unstructured residues causing aggregation at room temperature, was biologically active in the sense that it could complement a knock-out of wildtype RbfA, although it did not act as a suppressor for a 16S cold-sensitive mutation (C23U), nor did it interact stably with the 30S subunit. To complement this work, we report the 1H, 13C, and 15 N backbone and sidechain NMR resonance assignments of full length RbfA from Escherichia coli measured under physiological conditions (pH 7.6). This construct contains seven additional C-terminal residues from the cloning (i.e. one alanine and six residues from the HRV 3C cleavage site) and no aggregation issues were observed over a 1-week period at 293 K. The assignment data has been deposited in the BMRB data bank under Accession No. 27857.