Solution structure of the K50 class homeodomain PITX2 bound to DNA and implications for mutations that cause Rieger syndrome

We have determined the solution structure of a complex containing the K50 class homeodomain Pituitary homeobox protein 2 (PITX2) bound to its consensus DNA site (TAATCC). Previous studies have suggested that residue 50 is an important determinant of differential DNA-binding specificity among homeodomains. Although structures of several homeodomain-DNA complexes have been determined, this is the first structure of a native K50 class homeodomain. The only K50 homeodomain structure determined previously is an X-ray crystal structure of an altered specificity mutant, Engrailed Q50K (EnQ50K). Analysis of the NMR structure of the PITX2 homeodomain indicates that the lysine at position 50 makes contacts with two guanines on the antisense strand of the DNA, adjacent to the TAAT core DNA sequence, consistent with the structure of EnQ50K. Our evidence suggests that this side chain may make fluctuating interactions with the DNA, which is complementary to the crystal data for EnQ50K. There are differences in the tertiary structure between the native K50 structure and that of EnQ50K, which may explain differences in affinity and specificity between these proteins. Mutations in the human PITX2 gene are responsible for Rieger syndrome, an autosomal dominant disorder. Analysis of the residues mutated in Rieger syndrome indicates that many of these residues are involved in DNA binding, while others are involved in formation of the hydrophobic core of the protein. Overall, the role of K50 in homeodomain recognition is further clarified, and the results indicate that native K50 homeodomains may exhibit differences from altered specificity mutants.     

Engrailed and Hox homeodomain proteins contain a related Pbx interaction motif that recognizes a common structure present in Pbx.

Hox gene products have the ability to interact with either extradenticle or pbx gene products to bind cooperatively to DNA. The region in Hox proteins that is required for this interaction is located N-terminal of the homeodomain and contains a highly conserved hexapeptide. We now show that the engrailed gene products also contain a Pbx interaction motif positioned within a previously conserved region, the EH2 domain. The EH2 domain is located N-terminal of the homeodomain. Two tryptophan residues present in the Drosophila and murine Engrailed EH2 domain are required for cooperativity with extradenticle and Pbx, respectively. A second conserved domain, EH3, is required as well for cooperativity with Pbx, since deletions or an insertion in this region reduce cooperative DNA binding. Peptides containing the Pbx interaction motif of either Engrailed or Hox are capable of destabilizing Engrailed-Pbx and Hox-Pbx cooperative DNA binding. These data indicate that the Pbx interaction motifs present in Hox and engrailed gene products recognize a common structure present in the Pbx family of homeodomain proteins.     

An induction gene trap for identifying a homeoprotein-regulated locus

An important issue in developmental biology is the identification of homeoprotein target genes. We have developed a strategy based on the internalization and nuclear addressing of exogenous homeodomains, using an engrailed homeodomain (EnHD) to screen an embryonic stem (ES) cell gene trap library. Eight integrated gene trap loci responded to EnHD. One is within the bullous pemphigoid antigen 1 (BPAG1) locus, in a region that interrupts two neural isoforms. By combining in vivo electroporation with organotypic cultures, we show that an already identified BPAG1 enhancer/promoter is differentially regulated by homeoproteins Hoxc-8 and Engrailed in the embryonic spinal cord and mesencephalon. This strategy can therefore be used for identifying and mutating homeoprotein targets. Because homeodomain third helices can internalize proteins, peptides, phosphopeptides, and antisense oligonucleotides, this strategy should be applicable to other intracellular targets for characterizing genetic networks involved in a large number of physiopathological states.     

Investigation of Homeodomain Membrane Translocation Properties: Insights from the Structure Determination of Engrailed-2 Homeodomain in Aqueous and Membrane-Mimetic Environments

In addition to their well-known DNA-binding properties, homeodomains have the ability to efficiently translocate across biological membranes through still poorly-characterized mechanisms. To date, most biophysical studies addressing the mechanisms of internalization have focused on small synthetic peptides rather than full-length globular homeodomains. In this work, we characterized the conformational properties of chicken Engrailed 2 homeodomain (En2HD) in aqueous solution and in membrane mimetic environments using circular dichroism, Trp fluorescence, and NMR spectroscopy. En2HD adopts a well-defined three-helical bundle fold in aqueous solution. The Trp-48 residue, which is critical for internalization, is fully buried in the hydrophobic core. Circular dichroism and fluorescence reveal that a conformational transition occurs in anionic lipid vesicles and in micelles. En2HD loses its native three-dimensional structure in micellar environments but, remarkably, near-native helical secondary structures are maintained. Long-range interactions could be detected using site-directed spin labels, indicating that the three helices do not adopt extended orientations. Noncovalent paramagnetic probes yielded information about helix positioning and unveiled the burial of critical aromatic and basic residues within the micelles. Our results suggest that electrostatic interactions with membranes may be determinant in inducing a conformational change enabling Trp-48 to insert into membranes.     

Investigation of Homeodomain Membrane Translocation Properties: Insights from the Structure Determination of Engrailed-2 Homeodomain in Aqueous and Membrane-Mimetic Environments

In addition to their well-known DNA-binding properties, homeodomains have the ability to efficiently translocate across biological membranes through still poorly-characterized mechanisms. To date, most biophysical studies addressing the mechanisms of internalization have focused on small synthetic peptides rather than full-length globular homeodomains. In this work, we characterized the conformational properties of chicken Engrailed 2 homeodomain (En2HD) in aqueous solution and in membrane mimetic environments using circular dichroism, Trp fluorescence, and NMR spectroscopy. En2HD adopts a well-defined three-helical bundle fold in aqueous solution. The Trp-48 residue, which is critical for internalization, is fully buried in the hydrophobic core. Circular dichroism and fluorescence reveal that a conformational transition occurs in anionic lipid vesicles and in micelles. En2HD loses its native three-dimensional structure in micellar environments but, remarkably, near-native helical secondary structures are maintained. Long-range interactions could be detected using site-directed spin labels, indicating that the three helices do not adopt extended orientations. Noncovalent paramagnetic probes yielded information about helix positioning and unveiled the burial of critical aromatic and basic residues within the micelles. Our results suggest that electrostatic interactions with membranes may be determinant in inducing a conformational change enabling Trp-48 to insert into membranes.     

Molecular dynamics analysis of the engrailed homeodomain-DNA recognition

Molecular dynamics (MD) simulations were performed for investigating the role of Gln50 in the engrailed homeodomain-DNA recognition. Employing the crystal structure of free engrailed homeodomain and homeodomain-DNA complex as a starting structure, we carried out MD simulations of: (i) the complex between engrailed homeodomain and a 20 base-pair DNA containing TAATTA core sequence; (ii) the free engrailed homeodomain. The simulations show that homeodomain flexibility does not depend on its ligation state. The engrailed homeodomain shows similar flexibility, and the recognition helix-3 shows very similar characteristic of high rigidity and limited conformational space in two complexation states. At the same time, DNA structure has also no obvious conformational fluctuations. These results preclude the possibility of the side chain of Gln50 forming direct hydrogen bonds to the core DNA bases. MD simulations confirm a few well-conserved sites for water-mediated hydrogen bonds from protein to DNA are occupied by water molecules, and Gln50 interacts with corresponding core DNA bases through water-mediated hydrogen bonds. So Gln50 plays a relatively modest role in determining the affinity and specificity of the engrailed homeodomain. In addition, the electrostatic interaction between homeodomain and phosphate backbone of the DNA is a main factor for N- and C-terminal arm becoming ordered upon DNA binding.     

A Correspondence Between Solution-State Dynamics of an Individual Protein and the Sequence and Conformational Diversity of its Family

Conformational ensembles are increasingly recognized as a useful representation to describe fundamental relationships between protein structure, dynamics and function. Here we present an ensemble of ubiquitin in solution that is created by sampling conformational space without experimental information using “Backrub” motions inspired by alternative conformations observed in sub-Angstrom resolution crystal structures. Backrub-generated structures are then selected to produce an ensemble that optimizes agreement with nuclear magnetic resonance (NMR) Residual Dipolar Couplings (RDCs). Using this ensemble, we probe two proposed relationships between properties of protein ensembles: (i) a link between native-state dynamics and the conformational heterogeneity observed in crystal structures, and (ii) a relation between dynamics of an individual protein and the conformational variability explored by its natural family. We show that the Backrub motional mechanism can simultaneously explore protein native-state dynamics measured by RDCs, encompass the conformational variability present in ubiquitin complex structures and facilitate sampling of conformational and sequence variability matching those occurring in the ubiquitin protein family. Our results thus support an overall relation between protein dynamics and conformational changes enabling sequence changes in evolution. More practically, the presented method can be applied to improve protein design predictions by accounting for intrinsic native-state dynamics.     

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.     

Critical roles of interactions among switch I-preceding residues and between switch II and its neighboring alpha-helix in conformational dynamics of the GTP-bound Ras family small GTPases

GTP-bound forms of Ras family small GTPases exhibit dynamic equilibrium between two interconverting conformations, "inactive" state 1 and "active" state 2. A great variation exists in their state distribution; H-Ras mainly adopts state 2, whereas M-Ras predominantly adopts state 1. Our previous studies based on comparison of crystal structures representing state 1 and state 2 revealed the importance of the hydrogen-bonding interactions of two flexible effector-interacting regions, switch I and switch II, with the γ-phosphate of GTP in establishing state 2 conformation. However, failure to obtain both state structures from a single protein hampered further analysis of state transition mechanisms. Here, we succeed in solving two crystal structures corresponding to state 1 and state 2 from a single Ras polypeptide, M-RasD41E, carrying an H-Ras-type substitution in residue 41, immediately preceding switch I, in complex with guanosine 5'-(β,γ-imido)triphosphate. Comparison among the two structures and other state 1 and state 2 structures of H-Ras/M-Ras reveal two new structural features playing critical roles in state dynamics; interaction of residues 31/41 (H-Ras/M-Ras) with residues 29/39 and 30/40, which induces a conformational change of switch I favoring its interaction with the γ-phosphate, and the hydrogen-bonding interaction of switch II with its neighboring α-helix, α3-helix, which induces a conformational change of switch II favoring its interaction with the γ-phosphate. The importance of the latter interaction is proved by mutational analyses of the residues involved in hydrogen bonding. These results define the two novel functional regions playing critical roles during state transition.     

Computational Modeling of Allosteric Regulation in the Hsp90 Chaperones: A Statistical Ensemble Analysis of Protein Structure Networks and Allosteric Communications

A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks.     

Structures of perfringolysin O suggest a pathway for activation of cholesterol-dependent cytolysins

Cholesterol-dependent cytolysins (CDCs), a large family of bacterial toxins, are secreted as water-soluble monomers and yet are capable of generating oligomeric pores in membranes. Previous work has demonstrated that large scale structural rearrangements occur during this transition but the detailed mechanism by which these changes take place remains a puzzle. Despite evidence of structural and functional couplings between domains 3 and 4, the crystal structure of the CDC, perfringolysin O (PFO), shows the two domains do not make direct contact. Here, we present crystal structures of PFO that demonstrate movements of domain 4 are sufficient to trigger conformational changes that are transmitted through the molecule to the distant domain 3. These coupled movements result in a loss of many contacts between domain 3 and rest of the molecule that would eventually lead to the exposure of transmembrane regions in preparation for membrane insertion. The structures reveal a detailed molecular pathway that may be the basis for the allosteric transition that occurs on initial membrane binding leading to the exposure of membrane-spanning regions in a domain distant from the initial site of interaction.