Mario R. Ermácora

Engineering a compact non-native state of intestinal fatty acid-binding protein

The last three C-terminal residues (129-131) of intestinal fatty acid-binding protein (IFABP) participate in four main-chain hydrogen bonds and two electrostatic interactions to sequentially distant backbone and side-chain atoms. To assess if these interactions are involved in the final adjustment of the tertiary structure during folding, we engineered an IFABP variant truncated at residue 128. An additional mutation, Trp-6-->Phe, was introduced to simplify the conformational analysis by optical methods. Although the changes were limited to a small region of the protein surface, they resulted in an IFABP with altered secondary and tertiary structure. Truncated IFABP retains some cooperativity, is monomeric, highly compact, and has the molecular dimensions and shape of the native protein. Our results indicated that residues 129-131 are part of a crucial conformational determinant in which several long-range interactions, essential for the acquisition of the native state, are established. This work suggests that carefully controlled truncation can populate equilibrium non-native states under physiological conditions. These non-native states hold a great promise as experimental models for protein folding.

Study on fatty acid binding by proteins in yeast. Dissimilar results in Saccharomyces cerevisiae and Yarrowia lipolytica

1. The presence of soluble proteins with fatty acid binding activity was investigated in cell-free extracts from Saccharomyces cerevisiae and Yarrowia lipolytica cultures. 2. No significant fatty acid binding by proteins was detected in S. cerevisiae, even when grown on a fatty acid-rich medium, thus indicating that such proteins are not essential to fatty acid metabolism. 3. An inducible fatty acid binding protein (K0.5 = 3-4 microM) was found in Y. lipolytica which had grown on a minimal medium with palmitate as the sole source of carbon and energy. 4. The relative molecular mass of this protein was 100,000 as inferred from Sephacryl S-200 gel filtration.

Structure of the Mature Ectodomain of the Human Receptor-type Protein-tyrosine Phosphatase IA-2

IA-2 (insulinoma-associated protein 2) is a protein-tyrosine phosphatase receptor located in secretory granules of neuroendocrine cells. Initially, it attracted attention due to its involvement in the autoimmune response associated to diabetes. Later it was found that upon exocytosis, the cytoplasmic domain of IA-2 is cleaved and relocated to the nucleus, where it enhances the transcription of the insulin gene. A concerted functioning of the whole receptor is to be expected. However, very little is known about the structure and function of the transmembrane and extracellular domains of IA-2. To address this issue, we solved the x-ray structure of the mature ectodomain of IA-2 (meIA-2) to 1.30Å resolution. The fold of meIA-2 is related to the SEA (sea urchin sperm protein, enterokinase, agrin)) domains of mucins, suggesting its participation in adhesive contacts to the extracellular matrix and providing clues on how this kind of molecule may associate and form homo- and heterodimers. Moreover, we discovered that meIA-2 is self-proteolyzed in vitro by reactive oxygen species, suggesting the possibility of a new shedding mechanism that might be significant in normal function or pathological processes. Knowledge of meIA-2 structure should facilitate the search of its possible ligands and molecular interactions.

Combined measurement of diabetes mellitus immunological markers: an assessment of its benefits in adult-onset patients.

The convenience of combining the measurement of antibodies to glutamic acid decru·boxylase (GADA), protein tyrosine phosphatase (IA-2A), and autoantibodies to insulin (IAA) in diabetic patients was assessed. We analysed 71 type l and 11 5 adult-onset diabetic patient. The latter were grouped into three categories according to the time of evolution to insulin dependence. The main findings were as follows: (i) in type I diabetes, the combined analysis of GADA and IA-2A showed a sensitivity of 87.4% and was not appreciably improved by adding IAA; (ii) out of 31 adults who required insulin immediately or wi thin the first two years of diagnosis, 41.9, 29.0, and 6.5% were positive for at least one, two or all three, and all three markers, respectively; GADA was the most prevalent (35.5%) and IA-2A the least represented (16.1%); (iii) 34 adult patients with slow evolution to insulin dependence bowed a completely different profile: 5.9% were GADA positive and 23.5% were IAA positive and no double or triple positivity was observed as all patients were IA-2A negative; and (iv) 50 type 2 patients who had not required insulin treatment showed a low incidence of GADA (4%) as the only marker present. We conclude that a combined double-antigen test for GADA and TA-2A is a useful strategy for prospective screening of type I diabetes. However, in adults, the profile of individual markers di scloses the course to insulin dependence. There fore, it seems advisable to measure the markers separately, to allow a better classification of these patients, and help define their treatment.

Engineered variants of human glutamic acid decarboxylase (GAD) and autoantibody epitope recognition

Of the two homologous forms of glutamic acid decarboxylase, GAD65 and GAD67, only GAD65 is a common target of autoimmunity. Epitope profiles of autoantibodies to GAD65 (GADA) in 140 type 1 diabetes, adult-onset diabetes mellitus (AODM), and thyroid diseases (TD) were studied. Probes were GAD65, GAD65/67 hybrids (displaying separately GAD65 residues 1-95, 96-444, and 445-585), delta GAD65 (a truncated GAD65 spanning residues 69-585), and GAD67. delta GAD65 and GAD65 detected 137 and 125 positive patients, respectively. The hybrids reacted with 113 sera and in 3 cases disclosed cryptic epitopes. Eighteen patients reacted with GAD67, indicating GAD65-GAD67 cross-reactivity. Most patients recognized both middle and C-terminal epitopes, had low reactivity against N-terminal epitopes, and seldom displayed reactivity limited to the N or C terminus. Compared with type 1 and AODM, TD patients showed a greater prevalence of multiple reactivity and higher incidence of GAD67 positivity.

Structural selection of a native fold by peptide recognition. Insights into the thioredoxin folding mechanism.

Thioredoxins (TRXs) are monomeric alpha/beta proteins with a fold characterized by a central twisted beta-sheet surrounded by alpha-helical elements. The interaction of the C-terminal alpha-helix 5 of TRX against the remainder of the protein involves a close packing of hydrophobic surfaces, offering the opportunity of studying a fine-tuned molecular recognition phenomenon with long-range consequences on the acquisition of tertiary structure. In this work, we focus on the significance of interactions involving residues L94, L99, E101, F102, L103 and L107 on the formation of the noncovalent complex between reduced TRX1-93 and TRX94-108. The conformational status of the system was assessed experimentally by circular dichroism, intrinsic fluorescence emission and enzymic activity; and theoretically by molecular dynamics simulations (MDS). Alterations in tertiary structure of the complexes, resulting as a consequence of site specific mutation, were also examined. To distinguish the effect of alanine scanning mutagenesis on secondary structure stability, the intrinsic helix-forming ability of the mutant peptides was monitored experimentally by far-UV CD spectroscopy upon the addition of 2,2,2-trifluoroethanol, and also theoretically by Monte Carlo conformational search and MDS. This evidence suggests a key role of residues L99, F102 and L103 on the stabilization of the secondary structure of alpha-helix 5, and on the acquisition of tertiary structure upon complex formation. We hypothesize that the transition between a partially folded and a native-like conformation of reduced TRX1-93 would fundamentally depend on the consolidation of a cooperative tertiary unit based on the interaction between alpha-helix 3 and alpha-helix 5.

Structure of armadillo ACBP: a new member of the acyl-CoA-binding protein family

The X-ray structure of the tetragonal form of apo acyl-CoA-binding protein (ACBP) from the Harderian gland of the South American armadillo Chaetophractus villosus has been solved. ACBP is a carrier for activated long-chain fatty acids and has been associated with many aspects of lipid metabolism. Its secondary structure is highly similar to that of the corresponding form of bovine ACBP and exhibits the unique flattened alpha-helical bundle (up-down-down-up) motif reported for animal, yeast and insect ACBPs. Conformational differences are located in loops and turns, although these structural differences do not suffice to account for features that could be related to the unusual biochemistry and lipid metabolism of the Harderian gland.

A structural appraisal of sterol carrier protein 2

Sterol Carrier Protein 2 (SCP2) has been associated with lipid binding and transfer activities. However, genomic, proteomic, and structural studies revealed that it is an ubiquitous domain of complex proteins with a variety functions in all forms of life. High-resolution structures of representative SCP2 domains are available, encouraging a comprehensive review of the structural basis for its success. Most SCP2 domains pertain to three major families and are frequently found as stand-alone or at the C-termini of lipid related peroxisomal enzymes, acetyltransferases causing bacterial resistance, and bacterial environmentally important sulfatases. We (1) analyzed the structural basis of the fold and the classification of SCP2 domains; (2) identified structure-determined sequence features; (3) compared the lipid binding cavity of SCP2 and other lipid binding proteins; (4) surveyed proposed mechanisms of SCP2 mediated lipid transfer between membranes; and (5) uncovered a possible new function of the SCP2 domain as a protein-protein recognition device.

Biophysical characterization of the membrane-proximal ectodomain of the receptor-type protein-tyrosine phosphatase phogrin.

The receptor-type protein-tyrosine phosphatase (RPTP) phogrin is localized at the membrane of secretory granules of pancreatic islet β-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 β-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.

Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase

Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure.