The AXH module: an independently folded domain common to ataxin-1 and HBP1.

Ataxin-1 (ATX1), a human protein responsible for spinocerebellar ataxia type 1 in humans, shares a region of homology, named AXH module, with the apparently unrelated transcription factor HBP1. Here, we describe the first characterisation of the AXH module in terms of its structural properties and stability. By producing protein constructs spanning the AXH modules of ATX1 and HBP1 and by comparing their properties, we have identified the minimal region sufficient for forming independently folded units (domains). Knowledge of the AXH domain boundaries allows us to map many of the interactions of ATX1 with other molecules onto the AXH module. We further show that the AXH of ATX1 is a dimerisation domain and is able to recognise RNA with the same nucleotide preference previously described for the full-length protein. AXH is therefore a novel protein-protein and RNA binding motif.

The N-terminus of the fragile X mental retardation protein contains a novel domain involved in dimerization and RNA binding.

Fragile X syndrome, the most common cause of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). The emerging picture is that FMRP is involved in repression of translation through a complex network of protein-protein and protein-RNA interactions. Very little structural information is, however, available for FMRP that could help to understand its function. In particular, no structural studies are available about the N-terminus of the protein, a highly conserved region which is involved in several molecular interactions. Here, we explore systematically the ability of the FMRP N-terminus to form independently folded units (domains). We produced deletion mutants and tested their fold and functional properties by mutually complementary biophysical and biochemical techniques. On the basis of our data, we conclude that the N-terminus contains a domain, that we named NDF, comprising the first 134 amino acids. Most interestingly, NDF comprises two copies of a newly identified Agenet motif. NDF is thermally stable and has a high content of beta structure. In addition to being able to bind to RNA and to recognize some of the FMRP interacting proteins, NDF forms stable dimers and is able to interact, although weakly, with the full-length protein. Our data provide conclusive evidence that NDF is a novel motif for protein-protein and protein-RNA interactions and contains a previously unidentified dimerization site.

A structural approach to understanding the iron-binding properties of phylogenetically different frataxins.

Friedreich's ataxia (FRDA), an autosomal recessive cardio- and neurodegenerative disease, is caused by low expression of frataxin, a small mitochondrial protein, encoded in the nucleus. At the biochemical level, the lack of frataxin leads to dysregulation of mitochondrial iron homeostasis and oxidative damage, which eventually causes neuronal death. It is, however, still unclear whether frataxin is directly involved in iron binding, since the yeast orthologue, but not the human protein, has been shown to form large aggregates in the presence of large iron excess. We have compared the properties of three proteins from the frataxin family--the bacterial CyaY from Escherichia coli, the yeast Yfh1 and human frataxin--as representative of organisms of increasing complexity. We show that the three proteins have the same fold but different thermal stabilities and iron-binding properties. While human frataxin has no tendency to bind iron, CyaY forms iron-promoted aggregates with a behaviour similar to that of yeast frataxin. However, aggregation can be competed by chelator agents or by ionic strength. At physiological salt conditions, almost no aggregation is observed. The design of mutants produced to identify the protein surface involved in iron-promoted aggregation allows us to demonstrate that the process is mediated by a negatively charged surface ridge. Mutation of three of these residues is sufficient to convert CyaY in a protein with properties similar to those of human frataxin. On the other hand, mutation of the exposed surface of the beta sheet, which contains most of the conserved residues, does not affect aggregation, suggesting that iron binding is a non-conserved part of a more complex cellular function of frataxins.

Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin.

BACKGROUND: Lesions in the gene for frataxin, a nuclear-encoded mitochondrial protein, cause the recessively inherited condition Friedreich's ataxia. It is thought that the condition arises from disregulation of mitochondrial iron homeostasis, with concomitant oxidative damage leading to neuronal death. Very little is, as yet, known about the biochemical function of frataxin. RESULTS: Here, we show that the mature form of recombinant frataxin behaves in solution as a monodisperse species that is composed of a 15-residue-long unstructured N terminus and an evolutionarily conserved C-terminal region that is able to fold independently. The structure of the C-terminal domain consists of a stable seven-stranded antiparallel beta sheet packing against a pair of parallel helices. The structure is compact with neither grooves nor cavities, features that are typical of iron-binding modules. Exposed evolutionarily conserved residues cover a broad area and all cluster on the beta-sheet face of the structure, suggesting that this is a functionally important surface. The effect of two clinically occurring mutations on the fold was checked experimentally. When the mature protein was titrated with iron, no tendency to iron-binding or to aggregation was observed. CONCLUSIONS: Knowledge of the frataxin structure provides important guidelines as to the nature of the frataxin binding partner. The absence of all the features expected for an iron-binding activity, the large conserved area on its surface and lack of evidence for iron-binding activity strongly support an indirect involvement of frataxin in iron metabolism. The effects of point mutations associated with Friedreich's ataxia can be rationalised by knowledge of the structure and suggest possible models for the occurrence of the disease in compound heterozygous patients.

A potential allosteric subsite generated by domain swapping in bovine seminal ribonuclease.

Bovine seminal ribonuclease (BS-RNase) is a peculiar member of the pancreatic-like ribonuclease superfamily endowed with unique biological functions. It has been shown that native BS-RNase is a mixture of two distinct dimeric forms. The most abundant form is characterised by the swapping of the N-terminal helix. Kinetic studies have shown that this dimer is allosterically regulated, whereas the minor component, in which no swapping occurs, exhibits typical Michaelian kinetics. In order to correlate the catalytic properties with the structural features of BS-RNase, we have determined the crystal structure of the BS-RNase swapping dimer complexed with uridylyl(2'-5')guanosine. The structure of the complex was refined to an R value of 0.189 at 1.9 A resolution. Surprisingly, the enzyme binds four dinucleotide molecules, all in a non-productive way. In the two active sites, the guanine base is located in the subsite that is specific for pyrimidines. This unusual binding has been observed also in complexes of RNase A with guanine-containing nucleotides (retro-binding). One of the two additional dinucleotide molecules bound to the enzyme is located on the surface of the protein in a pocket generated by crystal packing; the second was found in a cavity at the interface between the two subunits of the swapping dimer. There are indications that the interface site plays a role in the allosteric regulation exhibited by BS-RNase. This finding suggests that domain swapping may not merely be a mechanism that proteins adopt for the transition from a monomeric to oligomeric state but can be used to achieve modulations in catalytic function.

Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains.

FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first approximately 400 residues, whereas the C-terminus provides a nonspecific binding surface.

Novel RNA-binding motif: the KH module.

The KH motif has recently been identified in single or multiple copies in a number of RNA associated proteins. Here we review the current knowledge accumulated about the sequence, structure, and functions of the KH. The multidomain architecture of most of the KH-containing proteins inspired an approach based on the production of peptides spanning the sequence of an isolated KH motif. Correct identification of the minimal length necessary for producing a folded peptide has had a number of important consequences for interpreting functional data. The presence of the KH motifs in fmr1, the protein responsible for the fragile X syndrome, and their possible role in the fmr1 functions are also discussed.

Binding of a substrate analog to a domain swapping protein: X-ray structure of the complex of bovine seminal ribonuclease with uridylyl(2',5')adenosine.

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. The native enzyme is a mixture of two dimeric forms with distinct structural features. The most abundant form is characterized by the swapping of N-terminal fragments. In this paper, the crystal structure of the complex between the swapping dimer and uridylyl(2',5')adenosine is reported at 2.06 A resolution. The refined model has a crystallographic R-factor of 0.184 and good stereochemistry. The quality of the electron density maps enables the structure of both the inhibitor and active site residues to be unambiguously determined. The overall architecture of the active site is similar to that of RNase A. The dinucleotide adopts an extended conformation with the pyrimidine and purine base interacting with Thr45 and Asn71, respectively. Several residues (Gln11, His12, Lys41, His119, and Phe120) bind the oxygens of the phosphate group. The structural similarity of the active sites of BS-RNase and RNase A includes some specific water molecules believed to be relevant to catalytic activity. Upon binding of the dinucleotide, small but significant modifications of the tertiary and quaternary structure of the protein are observed. The ensuing correlation of these modifications with the catalytic activity of the enzyme is discussed.

BS-RNase tetramers: an example of domain-swapped oligomers.

In the ribonuclease superfamily, dimericity is a unique feature of bovine seminal RNase (BS-RNase). In about two-thirds of native BS-RNase molecules, the two subunits interchange their N-terminal tails, thus generating domain-swapped dimers (MxM), which mostly responsible for enzyme biological activities and allostericity. Higher molecular weight BS-RNase oligomers can also be prepared [Libonati, M. (1969) Ital. J. Biochem. 18, 407-417.]. This paper reports on BS-RNase tetrameric derivatives which were isolated and enzymatically characterized. The data collected and the analysis of the crystal packing of MxM dimers suggested a structural model for tetramer assembly, in which the four subunits are enchained by multiple domain-swapping events.