Nina Pastor

The Structure and Function of Frataxin

ABSTRACT Frataxin, a highly conserved protein found in prokaryotes and eukaryotes, is required for efficient regulation of cellular iron homeostasis. Humans with a frataxin deficiency have the cardio- and neurodegenerative disorder Friedreichs ataxia, commonly resulting from a GAA trinucleotide repeat expansion in the frataxin gene. While frataxins specific function remains a point of controversy, the general consensus is that the protein assists in controlling cellular iron homeostasis by directly binding iron. This review focuses on the structural and biochemical aspects of iron binding by the frataxin orthologs and outlines molecular attributes that may help explain the proteins role in different cellular pathways.

Does TATA matter? A structural exploration of the selectivity determinants in its complexes with TATA box-binding protein

The binding of the TATA box-binding protein (TBP) to a TATA sequence in DNA is essential for eukaryotic basal transcription. TBP binds in the minor groove of DNA, causing a large distortion of the DNA helix. Given the apparent stereochemical equivalence of AT and TA basepairs in the minor groove, DNA deformability must play a significant role in binding site selection, because not all AT-rich sequences are bound effectively by TBP. To gain insight into the precise role that the properties of the TATA sequence have in determining the specificity of the DNA substrates of TBP, the solution structure and dynamics of seven DNA dodecamers have been studied by using molecular dynamics simulations. The analysis of the structural properties of basepair steps in these TATA sequences suggests a reason for the preference for alternating pyrimidine-purine (YR) sequences, but indicates that these properties cannot be the sole determinant of the sequence specificity of TBP. Rather, recognition depends on the interplay between the inherent deformability of the DNA and steric complementarity at the molecular interface.

Binding Mechanisms of TATA Box-Binding Proteins: DNA Kinking is Stabilized by Specific Hydrogen Bonds

One of the common mechanisms of DNA bending by minor groove-binding proteins is the insertion of protein side chains between basepair steps, exemplified in TBP (TATA box-binding protein)/DNA complexes. At the central basepair step of the TATA box TBP produces a noticeable decrease in twist and an increase in roll, while engaging in hydrogen bonds with the bases and sugars. This suggests a mechanism for the stabilization of DNA kinks that was explored here with ab initio quantum mechanical calculations and molecular dynamics/potential of mean force calculations. The hydrogen bonds are found to contribute the energy necessary to drive the conformational transition at the central basepair step. The Asn, Thr, and Gly residues involved in hydrogen bonding to the DNA bases and sugar oxygens form a relatively rigid motif in TBP. The interaction of this motif with DNA is found to be responsible for inducing the untwisting and rolling of the central basepair step. Notably, direct readout is shown not to be capable of discriminating between AA and AT steps, as the strength of the hydrogen bonds between TBP and the DNA are the same for both sequences. Rather, the calculated free energy cost for an equivalent conformational transition is found to be sequence-dependent, and is calculated to be higher for AA steps than for AT steps.

TIT for TAT: the properties of inosine and adenosine in TATA box DNA.

The sequence dependent conformation, flexibility and hydration properties of DNA molecules constitute selectivity determinants in the formation of protein-DNA complexes. TATA boxes in which AT basepairs (bp) have been substituted by IC bp (TITI box) allow for probing these selectivity determinants for the complexation with the TATA box-binding protein (TBP) with different sequences but identical chemical surfaces. The reference promoter Adenovirus 2 Major Late Promoter (mlp) is formed by the apposition of two sequences with very different dynamic properties: an alternating TATA sequence and an A-tract. For a comparative study, we carried out molecular dynamics simulations of two DNA oligomers, one containing the mlp sequence (2 ns), and the other an analog where AT basepairs were substituted by IC basepairs (1 ns). The simulations, carried out with explicit solvent and counterinons, yield straight purine tracts, the A-tract being stiffer than the I-tract, an alternating structure for the YRYR tracts, and hydration patterns that differ between the purine tracts and the alternating sequence tracts. A detailed analysis of the proposed interactions responsible for the stiffness of the purine tracts indicates that the stacking between the bases bears the strongest correlation to stiffness. The hydration properties of the minor groove in the two oligomers are distinctly different. Such differences are likely to be responsible for the stronger binding of TBP to mlp over the inosine-substituted variant. The calculations were made possible by the development, described here, of a new set of forcefield parameters for inosine that complement the published CHARMM all-hydrogen nucleic acid parametrization.

On the existence of very large nonadditivities for clusters of distorted water molecules

We present a detailed analysis, both at the self‐consistent field and electronic correlation levels, of the many‐body expansion of water clusters occurring with intramolecular relaxation. The results show extremely large nonadditivities, which lead to the proposal of an alternative many‐body expansion that shows convergence. From the results, it is clear that there is substantial intra–intermolecular coupling, an important contribution of the correlation energy to the stability of these clusters, and a crucial role of cooperativity in the stability of the condensed phases of water. It is also clear that these refinements should be included in water models attempting to simulate this substance.

Selective Binding of the TATA Box-Binding Protein to the TATA Box-Containing Promoter: Analysis of Structural and Energetic Factors

We report the results of an energy-based exploration of the components of selective recognition of the TATA box-binding protein (TBP) to a TATA box sequence that includes 1) the interaction between the hydrophobic Leu, Pro, and Phe residues of TBP with the TA, AT, AA, TT, and CG steps, by ab initio quantum mechanical calculations; and 2) the free energy penalty, calculated from molecular dynamics/potential of mean force simulations, for the conformational transition from A-DNA and B-DNA into the TA-DNA form of DNA observed in a complex with TBP. The GTAT, GATT, GAAT, and GTTT tetramers were explored. The results show that 1) the discrimination of TA, AT, AA, TT, or CG steps by TBP cannot rest on their interaction with the inserting Phe side chains; 2) the steric clash between the bulky and hydrophobic Pro and Leu residues and the protruding -NH2 group of guanine is responsible for the observed selectivity against any Gua-containing basepair; 3) the Pro and Leu residues cannot selectively discriminate among TA, AT, AA, or TT steps; and 4) the calculated energy required to achieve the TA-DNA conformation of DNA that is observed in the complex with TBP appears to be a key determinant for the observed selectivity against the AT, AA, and TT steps. The simulations also indicate that only the TA step can form a very efficient interbase hydrogen bond network in the TA-DNA conformation. Such an energetically stabilizing network is not achievable in the AA and TT steps. While it is viable in the AT step, structural constraints render the hydrogen bonding network energetically ineffective there.

PcExl1 a novel acid expansin-like protein from the plant pathogen Pectobacterium carotovorum, binds cell walls differently to BsEXLX1.

Microbial expansins act on plant cell walls similarly to plant expansins, albeit their loosening activity levels are tenfold lesser compared to plant expansins. We report the characterization of an expansin-like gene from the plant pathogen Pectobacterium carotovorum, named exl1. PcExl1 is an acidic protein that binds cellulose (Avicel), and weakens filter paper. The acidic nature of PcExl1 confers different binding properties when compared to Bacillus subtilis BsEXLX1, which is a basic protein. PcExl1 binding to wheat cell wall increased when acidic components were depleted, reaching a similar level to the binding to Avicel, indicating that cellulose is the target of PcExl1.

Use of in silico (computer) methods to predict and analyze the tertiary structure of plant hemoglobins.

Amino acid sequences for more than 60 plant hemoglobins (Hbs) are deposited in databases, but the tertiary structure of only 4 plant Hbs have been reported; thus, the gap between the reported sequences and structures of plant Hbs is large. Elucidating the structure of plant Hbs is essential to fully understanding the function of these proteins in plant cells. Determining the actual protein structure by experimental methods (i.e., by X-ray crystallography) requires considerable protein material and is expensive; thus, this type of work is limited to few laboratories around the world. In silico (computer) methods to predict the tertiary structure of proteins from amino acid sequences have been implemented and are helping reduce the sequence-structure gap. Thus, in silico methods are useful tools for predicting the tertiary structure of several plant Hbs from amino acid sequences deposited in databases. In this chapter, we describe a method for predicting and analyzing the structure of a rice Hb2 from the template structure of native rice Hb1. This method is based on a comparative modeling method that uses programs from the SWISS-MODEL server.

Progressive DNA Bending Is Made Possible by Gradual Changes in the Torsion Angle of the Glycosyl Bond

Structural comparisons have led to the suggestion that the conformational rearrangement that would be required to change A-DNA into the TA-DNA form of DNA observed in the complex with the TATA box binding protein (TBP) could be completed by modifying only the value of the glycosyl bond chi by approximately 45 degrees. The lack of a high number of crystal structures of this type makes it difficult to conclude whether a smooth transition from A-DNA to TA-DNA can occur without disrupting at any point either the Watson-Crick base pairing or the A-DNA conformation of the backbone. To explore the possibility of such a smooth transition, constrained molecular dynamics simulations were carried out for the double-stranded dodecamer d(GGTATATAAAAC), in which a transition from A-DNA to TA-DNA was induced by modifying only the chi angle values. The results demonstrate the feasibility of a continuous path in the A-DNA to TA-DNA transition. Varying extents of DNA curvature are also attainable, by maintaining the A-DNA backbone structure and Watson-Crick hydrogen bonding while changing the chi angle value smoothly from that in A-DNA to one corresponding to B-DNA.

Inhibition of Light Chain 6aJL2-R24G Amyloid Fiber Formation Associated with Light Chain Amyloidosis

Light chain amyloidosis (AL) is a deadly disease characterized by the deposition of monoclonal immunoglobulin light chains as insoluble amyloid fibrils in different organs and tissues. Germ line λ VI has been closely related to this condition; moreover, the R24G mutation is present in 25% of the proteins of this germ line in AL patients. In this work, five small molecules were tested as inhibitors of the formation of amyloid fibrils from the 6aJL2-R24G protein. We have found by thioflavin T fluorescence and transmission electron microscopy that EGCG inhibits 6aJL2-R24G fibrillogenesis. Furthermore, using nuclear magnetic resonance spectroscopy, dynamic light scattering, and isothermal titration calorimetry, we have determined that the inhibition is due to binding to the protein in its native state, interacting mainly with aromatic residues.