Insights into Lou Gehrig's disease from the structure and instability of the A4V mutant of human Cu,Zn superoxide dismutase

We have engineered enhanced DNA-binding function into the a1 homeodomain by making changes in a loop distant from the DNA-binding surface. Comparison of the free and bound a1 structures suggested a mechanism linking van der Waals stacking changes in this loop to the ordering of a final turn in the DNA-binding helix of a1. Inspection of the protein sequence revealed striking differences in amino acid identity at positions 24 and 25 compared to related homeodomain proteins. These positions lie in the loop connecting helix-1 and helix-2, which is involved in heterodimerization with the alpha 2 protein. A series of single and double amino acid substitutions (a1-Q24R, a1-S25Y, a1-S25F and a1-Q24R/S25Y) were engineered, expressed and purified for biochemical and biophysical study. Calorimetric measurements and HSQC NMR spectra confirm that the engineered variants are folded and are equally or more stable than the wild-type a1 homeodomain. NMR analysis of a1-Q24R/S25Y demonstrates that the DNA recognition helix (helix-3) is extended by at least one turn as a result of the changes in the loop connecting helix-1 and helix-2. As shown by EMSA, the engineered variants bind DNA with enhanced affinity (16-fold) in the absence of the alpha 2 cofactor and the variant alpha 2/a1 heterodimers bind cognate DNA with specificity and affinity reflective of the enhanced a1 binding affinity. Importantly, in vivo assays demonstrate that the a1-Q24R/S25Y protein binds with fivefold greater affinity than wild-type a1 and is able to partially suppress defects in repression by alpha 2 mutants. As a result of these studies, we show how subtle differences in residues at a surface distant from the functional site code for a conformational switch that allows the a1 homeodomain to become active in DNA binding in association with its cofactor alpha 2.     

Structure of a HoxB1-Pbx1 heterodimer bound to DNA: role of the hexapeptide and a fourth homeodomain helix in complex formation

Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. We report here the 2.35 A structure of a ternary complex containing a human HoxB1-Pbx1 heterodimer bound to DNA. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three-amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional alpha helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. The structure suggests a model for modulation of Hox DNA binding activity by Pbx1 and related proteins.     

Recognition of a DNA operator by a dimer composed of two different homeodomain proteins.

The yeast homeodomain proteins a1 and alpha 2 interact to form a heterodimer that binds DNA with high specificity. The DNA recognition element consists of two similar half sites, arranged with dyad symmetry and separated by a fixed number of base pairs. We demonstrate that in the a1 alpha 2-DNA complex, one of these half-sites is bound by a1 while the other is bound by alpha 2. These assignments allow a comparison of the chemical and nuclease protection patterns produced by both proteins when bound together to the hsg operator. Contrary to simple expectations, we propose that the a1 and alpha 2 homeodomains are arranged on the DNA in tandem, despite the fact that the recognition sequence is dyad symmetric.     

Lock and key binding of the HOX YPWM peptide to the PBX homeodomain

HOX homeodomain proteins bind short core DNA sequences to control very specific developmental processes. DNA binding affinity and sequence selectivity are increased by the formation of cooperative complexes with the PBX homeodomain protein. A conserved YPWM motif in the HOX protein is necessary for cooperative binding with PBX. We have determined the structure of a PBX homeodomain bound to a 14-mer DNA duplex. A relaxation-optimized procedure was developed to measure DNA residual dipolar couplings at natural abundance in the 20-kDa binary complex. When the PBX homeodomain binds to DNA, a fourth alpha-helix is formed in the homeodomain. This helix rigidifies the DNA recognition helix of PBX and forms a hydrophobic binding site for the HOX YPWM peptide. The HOX peptide itself shows some structure in solution and suggests that the interaction between PBX and HOX is an example of "lock and key" binding. The NMR structure explains the requirement of DNA for the PBX-HOX interaction and the increased affinity of DNA binding.     

Altering the DNA-binding specificity of the yeast Matalpha 2 homeodomain protein.

Homeodomain proteins are a highly conserved class of DNA-binding proteins that are found in virtually every eukaryotic organism. The conserved mechanism that these proteins use to bind DNA suggests that there may be at least a partial DNA recognition code for this class of proteins. To test this idea, we have investigated the sequence-specific requirements for DNA binding and repression by the yeast alpha2 homeodomain protein in association with its cofactors, Mcm1 and Mata1. We have determined the contribution for each residue in the alpha2 homeodomain that contacts the DNA in the co-crystal structures of the protein. We have also engineered mutants in the alpha2 homeodomain to alter the DNA-binding specificity of the protein. Although we were unable to change the specificity of alpha2 by making substitutions at residues 47, 54, and 55, we were able to alter the DNA-binding specificity by making substitutions at residue 50 in the homeodomain. Since other homeodomain proteins show similar changes in specificity with substitutions at residue 50, this suggests that there is at least a partial DNA recognition code at this position.     

NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.     

The binding sites for competitive antagonistic, allosteric antagonistic, and agonistic antibodies to the I domain of integrin LFA-1

We explore the binding sites for mAbs to the alpha I domain of the integrin alphaLbeta2 that can competitively inhibit, allosterically inhibit, or activate binding to the ligand ICAM-1. Ten mAbs, some of them clinically important, were mapped to species-specific residues. The results are interpreted with independent structures of the alphaL I domain determined in seven different crystal lattices and in solution, and which are present in three conformational states that differ in affinity for ligand. Six mAbs bind to adjacent regions of the beta1-alpha1 and alpha3-alpha4 loops, which show only small (mean, 0.8 angstroms; maximum, 1.8 angstroms) displacements among the eight I domain structures. Proximity to the ligand binding site and to noncontacting portions of the ICAM-1 molecule explains competitive inhibition by these mAbs. Three mAbs bind to a segment of seven residues in the beta5-alpha6 loop and alpha6 helix, in similar proximity to the ligand binding site, but on the side opposite from the beta1-alpha1/alpha3-alpha4 epitopes, and far from noncontacting portions of ICAM-1. These residues show large displacements among the eight structures in response to lattice contacts (mean, 3.6 angstroms; maximum, 9.4 angstroms), and movement of a buried Phe in the beta5-alpha6 loop is partially correlated with affinity change at the ligand binding site. Together with a lack of proximity to noncontacting portions of ICAM-1, these observations explain variation among this group of mAbs, which can either act as competitive or allosteric antagonists. One agonistic mAb binds distant from the ligand binding site of the I domain, to residues that show little movement (mean, 0.5 angstroms; maximum, 1.0 angstroms). Agonism by this mAb is thus likely to result from altering the orientation of the I domain with respect to other domains within an intact integrin alphaLbeta2 heterodimer.     

HU and IHF, two homologous histone-like proteins of Escherichia coli, form different protein-DNA complexes with short DNA fragments.

Using the gel retardation technique we have studied the protein-DNA complexes formed between HU--the major histone-like protein of Escherichia coli--and short DNA fragments. We show that several HU heterodimers bind DNA in a regularly spaced fashion with each heterodimer occupying about 9 base pairs. The alpha 2 and beta 2 HU homodimers form the same structure as the alpha beta heterodimer on double stranded DNA. However when compared to the heterodimer, they bind single stranded DNA with higher affinity. We also show that HU and the Integration Host Factor of E. coli (IHF) form different structures with the same DNA fragments. Moreover, HU seems to enhance the DNA-binding capacity of IHF to a DNA fragment which does not contain its consensus sequence.     

Arm-domain interactions can provide high binding cooperativity

Peptidyl arms extending from one protein domain to another protein domain mediate many important interactions in biology. A well-studied example of this type of protein-protein interaction occurs between the yeast homeodomain proteins, MAT alpha2 and MAT a1, which form a high-affinity heterodimer on DNA. The carboxyl-terminal arm extending from MAT alpha2 to MAT a1 has been proposed to produce an allosteric conformational change in the a1 protein that generates a very large increase in the DNA binding affinity of a1. Although early studies lent some support to this model, a more recent crystal structure determination of the free a1 protein argues against any allosteric change. This note presents a thermodynamic argument that accounts for the proteins' binding behavior, so that allosteric conformational changes are not required to explain the large affinity increase. The analysis presented here should be useful in analyzing binding behavior in other systems involving arm interactions.     

Characterization of the interaction interface and conformational dynamics of human TGIF1 homeodomain upon the binding of consensus DNA

The TG interacting factor-1 homeodomain (TGIF1-HD) binds with the consensus DNA motif 5′-TGTCA-3′ in gene promoters through its three-amino acid loop extension (TALE) type homeodomain, and then recruits co-regulators to regulate gene expression. Although the solution NMR structure of human TGIF1-HD has been reported previously, little is known about its DNA binding mechanism. NMR titrations have been extensively used to study mechanisms of ligand binding to target proteins; however, an intermediate exchange occurred predominantly between TGIF1-HD in the free and bound states when titrated with the consensus DNA, which resulted in poor-quality NMR spectra and precluded further exploration of its interaction interface and conformational dynamics. Here, the helix α3 of TGIF1-HD was speculated as the specific DNA binding interface by hydrogen–deuterium exchange mass spectrometry (HDX-MS) experiments, and subsequently confirmed by chemical exchange saturation transfer (CEST) spectroscopy. In addition, simultaneous conformational changes in other regions, including α1 and α2, were induced by DNA binding, explaining the observation of chemical shift perturbations from extensive residues besides those located in α3. Further, low-populated DNA-bound TGIF1-HD undergoing a slow exchange at a rate of 130.2 ± 3.6 s⁻¹ was derived from the analysis of the CEST data, and two residues, R220 and R221, located in the middle of α3 were identified to be crucial for DNA binding. Our study provides structural and dynamic insights into the mechanisms of TGIF1-HD recognition of extensive promoter DNA.     

The dimerization domain of LFB1/HNF1 related transcription factors: a hidden four helix bundle?

LFB1/HNF1 alpha and LFB3/HNF1 beta bind DNA as dimers and form heterodimers together in vivo and in vitro. The dimerization domain has been located in both proteins in the 32 N-terminal residues. In previous papers we have described the conformational stability as determined by CD and the secondary structure by NMR studies of a peptide with the amino acid sequence of the dimerization domain of LFB1/HNF1 alpha. This study presents a more complete characterization of similar synthetic peptides spanning the LFB3/HNF1 beta dimerization domain and the alpha/beta heterodimer. The HNF1 peptides represent an example of structures which cannot be determined by NOE data alone because they are not sufficient to define one unique solution. An approach is presented which combines NMR data, the protein structure database and structural analyses according to known principles of protein structure. On this basis we are able to determine possible solutions and to identify a four helix bundle as the structure most consistent with the experimental evidence.     

Distortion of the three-dimensional structure of the vnd/NK-2 homeodomain bound to DNA induced by an embryonically lethal A35T point mutation

The three-dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence was determined. This mutation to threonine from alanine in position 35 in helix II of the vnd/NK-2 homeodomain is associated with early embryonic lethality in Drosophila melanogaster. Although the unbound mutant protein is not structured, in the DNA-bound state it adopts the three-helix fold characteristic of all known homeodomains, but with alterations relative to the structure of the wild-type analogue. These structural modifications occur, and are accompanied by a 50-fold reduction in the DNA binding affinity, even though most of the protein-DNA interactions originally seen for the wild-type homeodomain are found likewise in the threonine analogue. Alterations include torsional angle changes in the loop between helix I and helix II, and in the turn between helix II and helix III, as well as in a distortion of the usual antiparallel orientation of helix I with respect to helix II. The alteration of the position of leucine 40 in the A35T mutant is proposed to explain the observed 1.27 ppm upfield shift of the corresponding amide proton resonance relative to the value observed for the wild-type analogue. A detailed comparison of the structures of the mutant A35T and wild-type vnd/NK-2 homeodomains bound to the cognate DNA is presented. The consequences of the structural alteration of the DNA-bound A35T mutant vnd/NK-2 protein may constitute the basis of the observed early embryonic lethality.     

Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution.

The 1:1 complex of the mutant Antp(C39----S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17,800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three-dimensional structure were based on the use of the fully 15N-labelled protein, two-dimensional homonuclear proton NOESY with 15N(omega 2) half-filter, and heteronuclear three-dimensional NMR experiments. Based on nearly complete sequence-specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H-1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix-turn-helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix-turn-helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39----S) homeodomain no defined spatial structure could be found for the apparently flexible N-terminal segment comprising residues 0-6.