An ancestral host defence peptide within human ß-defensin 3 recapitulates the antibacterial and antiviral activity of the full-length molecule.

Host defence peptides (HDPs) are critical components of innate immunity. Despite their diversity, they share common features including a structural signature, designated "γ-core motif". We reasoned that for each HDPs evolved from an ancestral γ-core, the latter should be the evolutionary starting point of the molecule, i.e. it should represent a structural scaffold for the modular construction of the full-length molecule, and possess biological properties. We explored the γ-core of human ß-defensin 3 (HBD3) and found that it: (a) is the folding nucleus of HBD3; (b) folds rapidly and is stable in human serum; (c) displays antibacterial activity; (d) binds to CD98, which mediates HBD3 internalization in eukaryotic cells; (e) exerts antiviral activity against human immunodeficiency virus and herpes simplex virus; and (f) is not toxic to human cells. These results demonstrate that the γ-core within HBD3 is the ancestral core of the full-length molecule and is a viable HDP per se, since it is endowed with the most important biological features of HBD3. Notably, the small, stable scaffold of the HBD3 γ-core can be exploited to design disease-specific antimicrobial agents.

Membrane protein 4F2/CD98 is a cell surface receptor involved in the internalization and trafficking of human ß-Defensin 3 in epithelial cells.

Human ß-defensins play a pivotal role in the innate immune response. Although expressed by and acting at epithelial surfaces, little is known about their specific interaction with epithelial structures. Here, we identify the transmembrane protein CD98 as a cell surface receptor involved in the internalization of human ß-defensin 3 (hBD3) in human epithelial A549 cells. CD98 and hBD3 extensively colocalize on the basolateral domain of A549. While verifying their direct binding by fluorescence resonance energy transfer and surface plasmon resonance, we mapped the interaction to CD98 residues 304-414, i.e. to the region known to interact with the proteins of intestinal bacteria during colonic invasion. Treatment of A549 cells with hBD3 dramatically reduces CD98 expression and conversely, knockdown of CD98 expression impairs hBD3 cell surface binding and internalization. Competition for bacterial binding to CD98 and downregulation of CD98 expression may represent novel mechanisms for the antibacterial activity of hBD3.

A novel prion protein-tyrosine hydroxylase interaction.

The prion protein (PrP) is currently one of the most studied molecules in the neurosciences. It is the main cause of a group of neurological diseases collectively called transmissible spongiform encephalopathies that severely affect both humans and a variety of mammals. Much effort has been directed to understanding the molecular basis of PrP activity, both in physiological and pathological terms. In this context, identification of neuronally-relevant interactors of PrP may play a crucial role. We recently discovered a specific, high-affinity (nanomolar KD) interaction with tyrosine hydroxylase (TH), a enzyme catalyzing the rate-limiting step in the synthesis of the neurotransmitter dopamine. Using molecular biological, biochemical and biophysical techniques we identified the C-terminal structured domain of PrP and the Nterminal regulatory domain of TH as interacting domains between these two proteins. This interaction does not affect TH activity in vitro, although co-expression experiments in HeLa and Chinese hamster ovary cells revealed that PrP is able to internalize TH. Moreover, TH modulated the level of expression of PrP and its localization at the plasma membrane. This novel interaction between two proteins of central importance in nervous system function may shed new light on our understanding of PrP in neurological diseases.

A novel anti-aldolase C antibody specifically interacts with residues 85-102 of the protein.

Aldolase C is a brain-specific glycolytic isozyme whose complete repertoire of functions are obscure. This lack of knowledge can be addressed using molecular tools that discriminate the protein from the homologous, ubiquitous paralog aldolase A. The anti-aldolase C antibodies currently available are polyclonal and not highly specific. We obtained the novel monoclonal antibody 9F against human aldolase C, characterized its isoform specificity and tested its performance. First, we investigated the specificity of 9F for aldolase C. Then, using bioinformatic tools coupled to molecular cloning and chemical synthesis approaches, we produced truncated human aldolase C fragments, and assessed 9F binding to these fragments by western blot and ELISA assays. This strategy revealed that residues 85-102 harbor the epitope-containing region recognized by 9F. The efficiency of 9F was demonstrated also for immunoprecipitation assays. Finally, surface plasmon resonance revealed that the protein has a high affinity toward the epitope-containing peptide. Taken together, our findings show that epitope recognition is sequence-driven and is independent of the three-dimensional structure. In conclusion, given its specific molecular interaction, 9F is a novel and powerful tool to investigate aldolase C's functions in the brain.

Cross-talk between prion protein and quadruplex-forming nucleic acids: a dynamic complex formation.

Prion protein (PrP) is involved in lethal neurodegenerative diseases, and many issues remain unclear about its physio-pathological role. Quadruplex-forming nucleic acids (NAs) have been found to specifically bind to both PrP cellular and pathological isoforms. To clarify the relevance of these interactions, thermodynamic, kinetic and structural studies have been performed, using isothermal titration calorimetry, surface plasmon resonance and circular dichroism methodologies. Three quadruplex-forming sequences, d(TGGGGT), r(GGAGGAGGAGGA), d(GGAGGAGGAGGA), and various forms of PrP were selected for this study. Our results showed that these quadruplexes exhibit a high affinity and specificity toward PrP, with K(D) values within the range 62÷630 nM, and a weaker affinity toward a PrP-ß oligomer, which mimics the pathological isoform. We demonstrated that the NA quadruplex architecture is the structural determinant for the recognition by both PrP isoforms. Furthermore, we spotted both PrP N-terminal and C-terminal domains as the binding regions involved in the interaction with DNA/RNAs, using several PrP truncated forms. Interestingly, a reciprocally induced structure loss was observed upon PrP-NA interaction. Our results allowed to surmise a quadruplex unwinding-activity of PrP, that may have a feedback in vivo.

Binding of methylene blue to a surface cleft inhibits the oligomerization and fibrillization of prion protein.

Neurodegenerative protein misfolding diseases, including prionopathies, share the common feature of accumulating specific misfolded proteins, with a molecular mechanism closely related. Misfolded prion protein (PrP) generates soluble oligomers that, in turn, aggregate into amyloid fibers. Preventing the formation of these entities, crucially associated with the neurotoxic and/or infectious properties of the resulting abnormal PrP, represents an attractive therapeutic strategy to ameliorate prionopathies. We focused our attention into methylene blue (MB), a well-characterized drug, which is under study against Alzheimer's disease and other neurodegenerative disorders. Here, we have undertaken an in vitro study on the effects of MB on oligomerization and fibrillization of human, ovine and murine PrP. We demonstrated that MB affects the kinetics of PrP oligomerization and reduces the amount of oligomer of about 30%, in a pH-dependent manner, by using SLS and DSC methodologies. Moreover, TEM images showed that MB completely suppresses fiber formation at a PrP:MB molar ratio of 1:2. Finally, NMR revealed a direct interaction between PrP and MB, which was mapped on a surface cleft including a fibrillogenic region of the protein. Our results allowed to surmise a mechanism of action in which the MB binding to PrP surface markedly interferes with the pathway towards oligomers and fibres. Therefore MB could be considered as a general anti-aggregation compound, acting against proteinopathies.

Stability against temperature of Sulfolobus solfataricus elongation factor 1 alpha, a multi-domain protein.

The elongation factors (EF-Tu/EF-1 alpha) are universal proteins, involved in protein biosynthesis. A detailed characterization of the stability against temperature of SsEF-1 alpha, a three-domain protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus is presented. Thermal denaturation of both the GDP-bound (SsEF-1 alpha*.GDP) and the ligand-free (nfSsEF-1 alpha) forms was investigated by means of circular dichroism and fluorescence measurements, over the 4.0-7.5 pH interval. Data indicate that the unfolding process is cooperative with no intermediate species and that the few inter-domain contacts identified in the crystal structure of SsEF-1 alpha play a role also at high temperatures. Finally, it is shown that the enzyme exhibits two different interchangeable thermally denatured states, depending on pH.

The effect of the osmolyte trimethylamine N-oxide on the stability of the prion protein at low pH.

A study of the effect of trimethylamine N-oxide on the stability of two recombinant forms of the prion protein PrP, an ovine full-length and a human truncated form, is here reported. Both thermal denaturation and denaturation at room temperature were analyzed at pH values above and below the pKa of trimethylamine N-oxide, which is close to 4.7. Surprisingly, results showed that not only is trimethylamine N-oxide able to decrease PrP thermal stability at low pH but it also acts as a strong denaturant at room temperature. Likely, this destabilization is due to the capability of the cationic form of trimethylamine N-oxide to interact with the protein backbone as well as to weaken electrostatic interactions which are important for PrP fold. These results constitute the first experimental measurement of the effect of trimethylamine N-oxide on PrP stability and provide an unambiguous proof of the destabilizing effect of this osmolyte on PrP at low pH.

Chemical denaturation of the elongation factor 1alpha isolated from the hyperthermophilic archaeon Sulfolobus solfataricus.

The stability against chemical denaturants of the elongation factor EF-1alpha (SsEF-1alpha), a protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus has been characterized in detail. Indeed, the atypical shape of the protein structure and the unusual living conditions of the host organism prompted us to analyze the effect of urea and guanidine hydrochloride (GuHCl) on the GDP complex of the enzyme (SsEF-1alpha x GDP) by fluorescence and circular dichroism. These studies were also extended to the nucleotide-free form of the protein (nfSsEF-1alpha). Interestingly, the experiments show that the denaturation curves of both SsEF-1alpha forms present a single inflection point, which is indicative of a cooperative unfolding process with no intermediate species. Moreover, the chemically induced unfolding process of both SsEF-1alpha x GDP and nfSsEF-1alpha is fully reversible. Both SsEF-1alpha forms exhibit remarkable stability against urea, but they do not display a strong resistance to the denaturing action of GuHCl. These findings suggest that electrostatic interactions significantly contribute to SsEF-1alpha stability.

Comparison of the crystallization and crystal packing of two Fab single-site mutant protein L complexes.

Protein L from Peptostreptococcus magnus (PpL) is a multidomain protein composed of four or five immunoglobulin-binding domains that target the kappa light chain of a large repertoire of human and murine antibodies. Thus, a single domain of this protein can be used to aid the crystallization of Fab, free or complexed to their antigen when it is not possible to obtain crystals without it. Each wild-type PpL domain has two light-chain binding sites that target the same region of the light chain and can thus bring together two Fab-antigen complexes within the crystal lattice. In this context the small PpL domain is sandwiched between two Fab and cannot participate in crystal contacts, thus mutants are unlikely to increase the chances of crystallizing a particular complex. However, it is possible to design mutants that can bind at only one site by making use of the crystal structures obtained so far. Such mutants will have a free surface that can participate in crystal contacts and that can be modified to improve its crystal contact-forming properties. Here, a comparison of two single-site mutants that differ at three different positions is reported. In both mutants two different tryptophan residues participate in crystal-packing interactions, suggesting that this residue may be particularly interesting for enhancing crystal-contact formation.

Denaturant-induced unfolding of the acetyl-esterase from Escherichia coli.

The stability of acetyl-esterase, Aes, from Escherichia coli against the denaturing action of urea and guanidine hydrochloride, GuHCl, has been investigated by means of circular dichroism and fluorescence measurements. The urea-induced unfolding curves show a single inflection point at 6.2 M urea, whereas the GuHCl-induced curves show two inflection points at 1.4 and 3.1 M GuHCl. The unfolding process is reversible with both urea and GuHCl. These results, together with similar experimental data on the mutant form V20D-Aes, suggest the presence of two domains in the Aes structure, which unfold more or less independently depending on the denaturant used. This is also supported by a 3D model obtained by homology modeling using the structure of brefeldine as a template. The effect of NaCl on the urea-induced unfolding curves of the enzyme has also been investigated.

Imino acids and collagen triple helix stability: characterization of collagen-like polypeptides containing Hyp-Hyp-Gly sequence repeats.

The analysis of factors contributing to the stability of proteins is a subject of intense debate. Particularly challenging is the study of structural proteins, since their function is their structure. Among these is collagen, the key structural component of bones, skin, cartilage, tendons, and other connecting tissues. It is well established that the collagen triple helix is characterized by the presence of hydroxyproline, whose content modulates triple helix thermal stability according to the requirement of the host organism. Because of the complexity and the fibrous nature of collagen, data on the stability and structure of this protein have been mainly obtained by the use of collagen-like polypeptides. On the basis of CD characterization of collagen-like polypeptides we here show that the presence of Hyp at the X position of repeating triplets Hyp-Hyp-Gly stabilizes the triple helix significantly. This extra-stabilization has been ascribed, by using molecular modeling, to the formation of a hydrogen bond between Hyp residues belonging to the X and the Y positions of adjacent chains. This communication also provides a comprehensive interpretation of the ensemble of available data on polypeptides containing proline derivatives.

Guanidine-induced unfolding of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus.

The unfolding induced by guanidine hydrochloride of the small protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus has been investigated by means of circular dichroism and fluorescence measurements. At neutral pH and room temperature the midpoint of the transition occurred at 4M guanidine hydrochloride. Thermodynamic information was obtained by means of both the linear extrapolation model and the denaturant binding model, in the assumption of a two-state N<==>D transition. A comparison with thermodynamic data determined from the thermal unfolding of Sso7d indicated that the denaturant binding model has to be preferred. Finally, it is shown that Sso7d is the most stable against both temperature and guanidine hydrochloride among a set of globular proteins possessing a very similar 3D structure.

Characterization of collagen-like heterotrimers: implications for triple-helix stability.

This article deals with the effects of proline hydroxylation on collagen triple-helix stability, an issue that is still under discussion. To investigate the structural determinants of triple-helix stabilization by hydroxyproline (Hyp), we here characterized spectroscopically triple-helix heterotrimers containing both chains of (Pro-Pro-Gly)10 and (Pro-Hyp-Gly)10. Results are discussed in relation to the various triple-helix stabilization mechanisms.

Effect of trifluoroethanol on the conformational stability of a hyperthermophilic esterase: a CD study.

The conformational stability of the hyperthermophilic esterase AFEST from Archeoglobus fulgidus against the denaturing action of 2,2,2-trifluoroethanol (TFE) has been investigated by means of circular dichroism (CD) measurements. At room temperature far-UV and near-UV CD spectra point out the occurrence of a co-operative transition from the native structure to a denatured state characterized by a high content of alpha-helix. The TFE concentration at half-completion of the transition proves to be 3.5 M (25% v v(-1)), by recording the molar ellipticity at both 222 and 276 nm. Thermal transition curves of AFEST in the absence and in the presence of TFE indicate a significant stability decrease on increasing the TFE concentration. The denaturation temperature is 99 degrees C for native AFEST, but becomes 85 degrees C at 1.4 M TFE (10% v v(-1)), and 56 degrees C at 2.8 M TFE (20% v v(-1)). It is also shown that, even though AFEST is very resistant to temperature, its resistance towards the denaturing action of TFE is similar to that of mesophilic proteins, including an esterase from Escherichia coli, AES. The proposal of a general mechanism for the TFE action on globular proteins leads to a reliable rationale of experimental data.

Thermal stability and DNA binding activity of a variant form of the Sso7d protein from the archeon Sulfolobus solfataricus truncated at leucine 54.

Sso7d is a 62-residue, basic protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Around neutral pH, it exhibits a denaturation temperature close to 100 degrees C and a non-sequence-specific DNA binding activity. Here, we report the characterization by circular dichroism and fluorescence measurements of a variant form of Sso7d truncated at leucine 54 (L54Delta). It is shown that L54Delta has a folded conformation at neutral pH and that its thermal unfolding is a reversible process, represented well by the two-state N <=> D transition model, with a denaturation temperature of 53 degrees C. Fluorescence titration experiments indicate that L54Delta binds tightly to calf thymus DNA, even though the binding parameters are smaller than those of the wild-type protein. Therefore, the truncation of eight residues at the C-terminus of Sso7d markedly affects the thermal stability of the protein, which nevertheless retains a folded structure and DNA binding activity.

Denaturing action of urea and guanidine hydrochloride towards two thermophilic esterases.

The stability of two thermophilic esterases, AFEST from Archaeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, against the denaturing action of urea and guanidine hydrochloride has been investigated by means of steady-state fluorescence and circular dichroism measurements. Experimental results indicate that the two enzymes, even though very resistant to temperature and urea, show a resistance to guanidine hydrochloride weaker than expected on the basis of data collected so far for a large set of globular proteins. Structural information available for AFEST and EST2 and ideas that emerged from studies on the molecular origin of the greater thermal stability of thermophiles allow the suggestion of a reliable rationale. The present results may be an indication that the optimization of charge-charge interactions on the protein surface is a key factor for the stability of the two esterases.

Temperature- and denaturant-induced unfolding of two thermophilic esterases.

We studied the temperature- and denaturant-induced denaturation of two thermophilic esterases, AFEST from Archeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, by means of circular dichroism measurements. Both enzymes showed a very high denaturation temperature: 99 degrees C for AFEST and 91 degrees C for EST2. They also showed a remarkable resistance against urea; at half-completion of the transition the urea concentration was 7.1 M for AFEST and 5.9 M for EST2. On the contrary, both enzymes showed a weak resistance against GuHCl; at half-completion of the transition the GuHCl concentration was 2.0 M for AFEST and 1.9 M for EST2. The thermodynamic parameters characterizing urea- and GuHCl-induced denaturation of the studied enzymes have been obtained by both the linear extrapolation model and the denaturant binding model. The dependence of the thermal stability on NaCl concentration for both esterases has also been determined. A careful analysis of the data, coupled with available structural information, has allowed the proposal of a reliable interpretation.