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SEA domains are ubiquitous in large proteins associated with highly glycosylated environments. Certain SEA domains undergo intramolecular proteolysis involving a nucleophilic attack of a serine hydroxyl group on the preceding glycine carbonyl. The mucin-1 (MUC1) SEA domain has been extensively investigated as a model of intramolecular proteolysis. Since neither a general base, a general acid, nor an oxyanion hole could be identified in MUC1 SEA, it has been suggested that proteolysis is accelerated by a non-planarity of the scissile peptide bond imposed by protein folding. A reactant distorted peptide bond has been also invoked to explain the autoproteolysis of several unrelated proteins. However, the only evidence of peptide distortion in MUC1 SEA stems from molecular dynamic simulations of the reactant modeled upon a single NMR structure of the cleaved product. We report the first high-resolution X-ray structure of cleaved MUC1 SEA. Structural comparison with uncleaved SEA domains suggests that the number of residues evolutionarily inserted in the cleaved loop of MUC1 SEA precludes the formation of a properly hydrogen-bonded beta turn. By sequence analysis, we show that this conformational frustration is shared by all known cleaved SEA domains. In addition, alternative conformations of the uncleaved precursor could be modeled in which the scissile peptide bond is planar. The implications of these structures for autoproteolysis are discussed in the light of the previous research on autoproteolysis.

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Protein dynamics, folding, and thermodynamics represent a central aspect of biophysical chemistry. pH, temperature, and denaturant perturbations inform our understanding of diverse contributors to stability and rates. In this work, we performed a thermodynamic analysis using a combined experimental and computational approach to gain insights into the role of electrostatics in the folding reaction of a psychrophile frataxin variant from Psychromonas ingrahamii. This folding reaction is strongly modulated by pH with a single, narrow, and well-defined transition state with ∼80% compactness, ∼70% electrostatic interactions, and ∼60% hydration shell compared to the native state (αD = 0.82, αH = 0.67, and αΔCp = 0.59). Our results are best explained by a two-proton/two-state model with very different pKa values of the native and denatured states (∼5.5 and ∼8.0, respectively). As a consequence, the stability strongly increases from pH 8.0 to 6.0 (|ΔΔG°| = 5.2 kcal mol-1), mainly because of a decrease in the TΔS°. Variation of ΔH° and ΔS° at pH below 7.0 is dominated by a change in ΔHf⧧ and ΔSf⧧, while at pH above 7.0, it is governed by ΔHu⧧ and ΔSu⧧. Molecular dynamics simulations showed that these pH modulations could be explained by the fluctuations of two regions, rich in electrostatic contacts, whose dynamics are pH-dependent and motions are strongly correlated. Results presented herein contribute to the understanding of the stability and dynamics of this frataxin variant, pointing to an intrinsic feature of the family topology to support different folding mechanisms.

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Local events that affect specific regions of proteins are of utmost relevance for stability and function. The aim of this study is to quantitatively assess the importance of locally-focused dynamics by means of a simple chemical modification procedure. Taking human Frataxin as a working model, we investigated local fluctuations of the C-terminal region (the last 16 residues of the protein) by means of three L → C replacement mutants: L98C, L200C and L203C. The conformation and thermodynamic stability of each variant was assessed. All the variants exhibited native features and high stabilities: 9.1 (wild type), 8.1 (L198C), 7.0 (L200C) and 10.0 kcal mol-1 (L203C). In addition, kinetic rates of Cys chemical modification by DTNB and DTDPy were measured, conformational dynamics data were extracted and free energy for the local unfolding of the C-terminal region was estimated. The analysis of these results indicates that the conformation of the C-terminal region fluctuates with partial independence from global unfolding events. Additionally, numerical fittings of the kinetic model of the process suggest that the local transition occurs in the seconds to minutes timescale. In fact, standard free energy differences for local unfolding were found to be significantly lower than those of the global unfolding reaction, showing that chemical modification results may not be explained in terms of the global unfolding reaction alone. These results provide unequivocal experimental evidence of local phenomena with global effects and contribute to understanding how global and local stability are linked to protein dynamics.

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Human frataxin (FXN) is a highly conserved mitochondrial protein involved in iron homeostasis and activation of the iron-sulfur cluster assembly. FXN deficiency causes the neurodegenerative disease Friedreich's Ataxia. Here, we investigated the effect of alterations in loop-1, a stretch presumably essential for FXN function, on the conformational stability and dynamics of the native state. We generated four loop-1 variants, carrying substitutions, insertions and deletions. All of them were stable and well-folded proteins. Fast local motions (ps-ns) and slower long-range conformational dynamics (µs-ms) were altered in some mutants as judged by NMR. Particularly, loop-1 modifications impact on the dynamics of a distant region that includes residues from the ß-sheet, helix α1 and the C-terminal. Remarkably, all the mutants retain the ability to activate cysteine desulfurase, even when two of them exhibit a strong decrease in iron binding, revealing a differential sensitivity of these functional features to loop-1 perturbation. Consequently, we found that even for a small and relatively rigid protein, engineering a loop segment enables to alter conformational dynamics through a long-range effect, preserving the native-state structure and important aspects of function.

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Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.

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Frataxin is an evolutionary conserved protein that participates in iron metabolism. Deficiency of this small protein in humans causes a severe neurodegenerative disease known as Friedreich's ataxia. A number of studies indicate that frataxin binds iron and regulates Fe-S cluster biosynthesis. Previous structural studies showed that metal binding occurs mainly in a region of high density of negative charge. However, a comprehensive characterization of the binding sites is required to gain further insights into the mechanistic details of frataxin function. In this work, we have solved the X-ray crystal structures of a cold-adapted frataxin from a psychrophilic bacterium in the presence of cobalt or europium ions. We have identified a number of metal-binding sites, mainly solvent exposed, several of which had not been observed in previous studies on mesophilic homologues. No major structural changes were detected upon metal binding, although the structures exhibit significant changes in crystallographic B-factors. The analysis of these B-factors, in combination with crystal packing and RMSD among structures, suggests the existence of localized changes in the internal motions. Based on these results, we propose that bacterial frataxins possess binding sites of moderate affinity for a quick capture and transfer of iron to other proteins and for the regulation of Fe-S cluster biosynthesis, modulating interactions with partner proteins.

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Phogrin/IA-2ß and ICA512/IA-2 are two paralogs receptor-type protein-tyrosine phosphatases (RPTP) that localize in secretory granules of various neuroendocrine cells. In pancreatic islet ß-cells, they participate in the regulation of insulin secretion, ensuring proper granulogenesis, and ß-cell proliferation. The role of their cytoplasmic tail has been partially unveiled, while that of their luminal region remains unclear. To advance the understanding of its structure-function relationship, the X-ray structure of the mature ectodomain of phogrin (ME phogrin) at pH 7.4 and 4.6 has been solved at 1.95- and 2.01-Å resolution, respectively. Similarly to the ME of ICA512, ME phogrin adopts a ferredoxin-like fold: a sheet of four antiparallel ß-strands packed against two α-helices. Sequence conservation among vertebrates, plants and insects suggests that the structural similarity extends to all the receptor family. Crystallized ME phogrin is monomeric, in agreement with solution studies but in striking contrast with the behavior of homodimeric ME ICA512. The structural details that may cause the quaternary structure differences are analyzed. The results provide a basis for building models of the overall orientation and oligomerization state of the receptor in biological membranes.

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The receptor-type protein-tyrosine phosphatase (RPTP) phogrin is localized at the membrane of secretory granules of pancreatic islet ß-cells and, similarly to the closely related ICA512, plays a role in the regulation of insulin secretion, in ensuring proper granulogenesis and stability, and in the regulation of ß-cell growth. The mature membraneproximal ectodomain of phogrin (MPE phogrin) was produced as a recombinant protein and characterized. CD, fluorescence, controlled proteolysis, size-exclusion chromatography, and multi-angle light scattering showed that it is a properlyfolded monomeric domain. Equilibrium experiments, in the presence of guanidinium chloride and thermal unfolding, suggest a two-state mechanism with a ΔG of 2.3-3.3 kcal/mol, respectively. The study establishes common features and differences of MPE phogrin and the homologous ectodomain of ICA512. A homology model of phogrin was built based in the x-ray structure of MPE ICA512. The model is a starting point for modeling the entire receptor and for testing the quaternary structure and interactions of this protein in vivo. A description of the membrane insertion mode and putative interacting surfaces of this large protein is fundamental for the understanding of its biological function.

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ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

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The three-dimensional structure of protein is encoded in the sequence, but many amino acid residues carry no essential conformational information, and the identity of those that are structure-determining is elusive. By circular permutation and terminal deletion, we produced and purified 25 Bacillus licheniformis beta-lactamase (ESBL) variants that lack 5-21 contiguous residues each, and collectively have 82% of the sequence and 92% of the non-local atom-atom contacts eliminated. Circular dichroism and size-exclusion chromatography showed that most of the variants form conformationally heterogeneous mixtures, but by measuring catalytic constants, we found that all populate, to a greater or lesser extent, conformations with the essential features of the native fold. This suggests that no segment of the ESBL sequence is essential to the structure as a whole, which is congruent with the notion that local information and modular organization can impart most of the tertiary fold specificity and cooperativity.

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