Biomedical subjects

Simona Miron

Publications and source records attributed to Simona Miron.

11 recordsLinked to original sources

PubMed2025

Moss BRCA2 lacking the canonical DNA-binding domain promotes homologous recombination and binds to DNA.

BRCA2 is crucial for mediating homology-directed DNA repair (HDR) through its binding to single-stranded DNA (ssDNA) and the recombinases RAD51 and DMC1. Most BRCA2 orthologs have a canonical DNA-binding domain (DBD) with the exception of Drosophila melanogaster. It remains unclear whether such a noncanonical BRCA2 variant without DBD possesses a DNA-binding activity. Here, we identify a new noncanonical BRCA2 in the model plant Physcomitrium patens (PpBRCA2). We establish that PpBRCA2 is essential for genome integrity maintenance, somatic DNA double-strand break (DSB) repair, HDR-mediated gene targeting, and RAD51 foci recruitment at DNA break sites. PpBRCA2 is also critical for DSB repair during meiosis. Interestingly, PpBRCA2 interacts strongly with RAD51 but weakly with DMC1, suggesting a distinct meiotic function compared to other BRCA2 homologs. Despite lacking the canonical DBD, PpBRCA2 binds ssDNA through its disordered N-terminal region and efficiently promotes HDR. Our work highlights that the ssDNA binding capacity of BRCA2 homologs is conserved regardless of the presence of a canonical DBD and provides a deeper understanding of BRCA2's functional diversity across species.

BRCA2 Protein↗

PubMed2006

Crystallization and preliminary X-ray diffraction data of the complex between human centrin 2 and a peptide from the protein XPC.

Centrins are highly conserved calcium-binding proteins involved in the nucleotide-excision repair pathway as a subunit of the heterotrimer including the XPC and hHR23B proteins. A complex formed by a Ca2+-bound human centrin 2 construct (the wild type lacking the first 25 amino acids) with a 17-mer peptide derived from the XPC sequence (residues Asn847-Arg863) was crystallized. Data were collected to 1.65 angstroms resolution from crystals grown in 30% monomethyl polyethylene glycol (MPEG) 500, 100 mM NaCl and 100 mM Bicine pH 9.0. Crystals are monoclinic and belong to space group C2, with two molecules in the asymmetric unit. The unit-cell parameters are a = 60.28, b = 59.42, c = 105.14 angstroms, alpha = gamma = 90, beta = 94.67 degrees. A heavy-atom derivative was obtained by co-crystallization with Sr2+. The substitution was rationalized by calorimetry experiments, which indicate a binding constant for Sr2+ of 4.0 x 10(4) M(-1).

Calcium-Binding Proteins↗

PubMed2006

Flexibility and plasticity of human centrin 2 binding to the xeroderma pigmentosum group C protein (XPC) from nuclear excision repair.

Human centrin 2 is a component of the nucleotide excision repair system, as a subunit of the heterotrimer including xeroderma pigmentosum group C protein (XPC) and hHR23B. The C-terminal domain of centrin (C-HsCen2) binds strongly a peptide from the XPC protein (P1-XPC: N(847)-R(863)). Here, we characterize the solution Ca(2+)-dependent structural and molecular features of the C-HsCen2 in complex with P1-XPC, mainly using NMR spectroscopy and molecular modeling. The N-terminal half of the peptide, organized as an alpha helix is anchored into a deep hydrophobic cavity of the protein, because of three bulky hydrophobic residues in position 1-4-8 and electrostatic contacts with the centrin helix E. Investigation of the whole centrin interactions shows that the N-terminal domain of the protein is not involved in the complex formation and is structurally independent from the peptide-bound C-terminal domain. The complex may exist in three different binding conformations corresponding to zero, one, and two Ca(2+)-bound states, which may exchange with various rates and have distinct structural stability. The various features of the intermolecular interaction presented here constitute a centrin-specific mode for the target binding.

Amino Acid Motifs↗

PubMed2006

The N-terminal domain of human centrin 2 has a closed structure, binds calcium with a very low affinity, and plays a role in the protein self-assembly.

Centrins are well-conserved calcium binding proteins from the EF-hand superfamily implicated in various cellular functions, such as centrosome duplication, DNA repair, and nuclear mRNA export. The intrinsic molecular flexibility and the self-association tendency make difficult the structural characterization of the integral protein. In this paper we report the solution structure, the Ca2+ binding properties, and the intermolecular interactions of the N-terminal domain of two human centrin isoforms, HsCen1 and HsCen2. In the absence of Ca2+, the N-terminal construct of HsCen2 revealed a compact core conformation including four almost antiparallel alpha-helices and a short antiparallel beta-sheet, very similar to the apo state structure of other calcium regulatory EF-hand domains. The first 25 residues show a highly irregular and dynamic structure. The three-dimensional model for the N-terminal domain of HsCen1, based on the high sequence conservation and NMR spectroscopic data, shows very close structural properties. Ca2+ titration of the apo-N-terminal domain of HsCen1 and HsCen2, monitored by NMR spectroscopy, revealed a very weak affinity (10(2)-10(3) M(-1)), suggesting that the cellular role of this domain is not calcium dependent. Isothermal calorimetric titrations showed that an 18-residue peptide, derived from the N-terminal unstructured fragment, has a significant affinity (approximately 10(5) M(-1)) for the isolated C-terminal domain, suggesting an active role in the self-assembly of centrin molecules.

Amino Acid Sequence↗

PubMed2005

New synchrotron radiation circular dichroism end-station on DISCO beamline at SOLEIL synchrotron for biomolecular analysis.

The novel Synchrotron Radiation Circular Dichroism (SRCD) technique is becoming a new tool of investigation for the molecular structures of biomolecules, like proteins, carbohydrates or others bio-materials. Here, we describe the characteristics of a new experimental end-station for circular dichroism studies, in construction on DISCO beamline at SOLEIL synchrotron (Saint-Aubin, France). This experimental end-station will be an open facility for the community of researchers in structural biology. In order to show the kind of information accessible with this type of technique, we give an example: the conformational study of the galactose mutarotase from Escherichia coli, an enzyme involved in the galactose metabolism. This study was made using an operational SRCD station available at SRS (Daresbury Laboratory, UK).

Amino Acid Sequence↗

PubMed2004

Transient aggregation and stable dimerization induced by introducing an Alzheimer sequence into a water-soluble protein.

Transient contacts between denatured polypeptide chains are likely to play an important part in the initial stages of protein aggregation and fibrillation. To analyze the nature of such contacts, we have carried out a protein engineering study of the 102-residue protein U1A, which aggregates transiently in the wild-type form during refolding from the guanidinium chloride-denatured state. We have prepared a series of mutants with increased aggregation tendencies by increasing the homology between two beta-strands of U1A and the Alzheimer peptide (beta-AP). These mutants undergo transient aggregation during refolding, as measured by concentration dependence, double-jump experiments, and binding of ANS, a probe for exposed hydrophobic patches on protein surfaces. The propensity to aggregate increases with increasing homology to beta-AP. Further, the degree of transient ANS binding correlates reasonably well with the structural parameters recently shown to play a role in the fibrillation of natively unfolded proteins. Two mutants highly prone to transient aggregation, U1A-J and U1A-G, were also studied by NMR. Secondary structural elements of the U1A-J construct (with lower beta-AP homology) are very similar to those observed in U1A-wt. In contrast, the high-homology construct U1A-G exhibits local unfolding of the C-terminal helix, which packs against the beta-sheet in the wild-type protein. U1A-G is mainly dimeric according to (15)N spin relaxation data, and the dimer interface most likely involves the beta-sheet. Our data suggest that the transient aggregate relies on specific intermolecular interactions mediated by structurally flexible regions and that contacts may be formed in different beta-strand registers.

Alzheimer Disease↗

PubMed2004

Structural and dynamic studies on ligand-free adenylate kinase from Mycobacterium tuberculosis revealed a closed conformation that can be related to the reduced catalytic activity.

Tuberculosis is the leading cause of death worldwide from a single infectious disease. Search of new therapeutic tools requires the discovery and biochemical characterization of new potential targets among the bacterial proteins essential for the survival and virulence. Among them are the nucleoside monophosphate kinases, involved in the nucleotide biosynthesis. In this work, we determined the solution structure of adenylate kinase (AK) from Mycobacterium tuberculosis (AKmt), a protein of 181 residues that was found to be essential for bacterial survival. The structure was calculated by a simulated annealing protocol and energy minimization using experimental restraints, collected by nuclear magnetic resonance spectroscopy. The final, well-defined 20 NMR structures show an average root-mean-square deviation of 0.77 A for the backbone atoms in regular secondary structure segments. The protein has a central CORE domain, composed of a five-stranded parallel beta-sheet surrounded by seven alpha-helices, and two peripheral domains, AMPbd and LID. As compared to other crystallographic structures of free form AKs, AKmt is more compact, with the AMP(bd) domain closer to the CORE of the protein. Analysis of the (15)N relaxation data enabled us to obtain the global rotational correlation time (9.19 ns) and the generalized order parameters (S(2)) of amide vectors along the polypeptide sequence. The protein exhibits restricted movements on a picosecond to nanosecond time scale in the secondary structural regions with amplitudes characterized by an average S(2)() value of 0.87. The loops beta1/alpha1, beta2/alpha2, alpha2/alpha3, alpha3/alpha4, alpha4/beta3, beta3/alpha5, alpha6/alpha7 (LID), alpha7/alpha8, and beta5/alpha9 exhibit rapid fluctuations with enhanced amplitudes. These structural and dynamic features of AKmt may be related to its low catalytic activity that is 10-fold lower than in their eukaryote counterparts.

Adenylate Kinase↗

PubMed2003

Xeroderma pigmentosum group C protein possesses a high affinity binding site to human centrin 2 and calmodulin.

Human centrin 2 (HsCen2), a member of the EF-hand superfamily of Ca2+-binding proteins, is commonly associated with centrosome-related structures. The protein is organized in two domains, each containing two EF-hand motifs, but only the C-terminal half exhibits Ca2+ sensor properties. A significant fraction of HsCen2 is localized in the nucleus, where it was recently found associated with the xeroderma pigmentosum group C protein (XPC), a component of the nuclear excision repair pathway. Analysis of the XPC sequence (940 residues), using a calmodulin target recognition software, enabled us to predict two putative binding sites. The binding properties of the two corresponding peptides were investigated by isothermal titration calorimetry. Only one of the peptides (P1-XPC) interacts strongly (Ka = 2.2 x 10(8) m-1, stoichiometry 1:1) with HsCen2 in a Ca2+-dependent manner. This peptide also binds, with a similar affinity (Ka = 1.1 x 10(8) m-1) to a C-terminal construct of HsCen2, indicating that the interaction with the integral protein is mainly the result of the contribution of the C-terminal half. The second peptide (P2-XPC) failed to show any detectable binding either to HsCen2 or to its C-terminal lobe. The two peptides interact with different affinities and mechanisms with calmodulin. Circular dichroism and nuclear magnetic resonance were used to structurally characterize the complex formed by the C-terminal domain of HsCen2 with P1-XPC.

Amino Acid Sequence↗

PubMed2003

C-terminal half of human centrin 2 behaves like a regulatory EF-hand domain.

Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer. Only the longer C-terminal domain, in the Ca(2+)-saturated state and in the presence of Na(+) ions, was amenable to structure determination by nuclear magnetic resonance. The solution structure of LC-HsCen2 reveals an open two EF-hand structure, similar to the conformation of related Ca(2+)-saturated regulatory domains. Unexpectedly, the N-terminal helix segment (F86-T94) lies over the exposed hydrophobic cavity. This unusual intramolecular interaction increases considerably the Ca(2+) affinity and constitutes a useful model for the target binding.

Amino Acid Sequence↗

PubMed2002

Calbindin D28k exhibits properties characteristic of a Ca2+ sensor.

Calbindin D(28k) is a member of the calmodulin superfamily of Ca(2+)-binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca(2+) buffer, but the studies presented in this work indicate that it may also act as a Ca(2+) sensor. The results show that Mg(2+) binds to the same sites as Ca(2+) with an association constant of approximately 1.4.10(3) m(-1) in 0.15 m KCl. The four high affinity sites in calbindin D(28k) bind Ca(2+) in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg(2+), the Ca(2+) affinity is reduced by a factor of 2, and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca(2+) concentration in a resting cell calbindin D(28k) is saturated to 40-75% with Mg(2+) but to less than 9% with Ca(2+). In contrast, the protein is expected to be nearly fully saturated with Ca(2+) at the Ca(2+) level of an activated cell. A substantial conformational change is observed upon Ca(2+) binding, but only minor structural changes take place upon Mg(2+) binding. This suggests that calbindin D(28k) undergoes Ca(2+)-induced structural changes upon Ca(2+) activation of a cell. Thus, calbindin D(28k) displays several properties that would be expected for a protein involved in Ca(2+)-induced signal transmission and hence may function not only as a Ca(2+) buffer but also as a Ca(2+) sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca(2+), becomes exposed upon Ca(2+) binding.

Amino Acid Sequence↗

PubMed2002

Structural and nucleotide-binding properties of YajQ and YnaF, two Escherichia coli proteins of unknown function.

Structural genomics is a new approach in functional assignment of proteins identified via whole-genome sequencing programs. Its rationale is that nonhomologous proteins performing similar or related biological functions might have similar tertiary structure. We used dye pseudoaffinity chromatography, two-dimensional gel electrophoresis, and mass spectrometry to identify two novel Escherichia coli nucleotide-binding proteins, YnaF and YajQ. YnaF exhibited significant sequence identity with MJ0577, an ATP-binding protein from a hyperthermophile (Methanococcus jannaschii), and with UspA, a protein from Haemophilus influenzae that belongs to the Universal Stress Protein family. YnaF conserves the ATP-binding site and the dimeric structure observed in the crystal of MJ0577. The protein YajQ, present in many bacterial genomes, is missing in eukaryotes. In the absence of significant similarities of YajQ to any solved structure, we determined its structural and ligand-binding properties by NMR and isothermal titration calorimetry. We demonstrate that YajQ is composed of two domains, each centered on a beta-sheet, that are connected by two helical segments. NMR studies, corroborated with local sequence conservation among YajQ homologs in various bacteria, indicate that one of the beta-sheets is mostly involved in biological activity.

Adenosine Triphosphate↗