Kinetic stability may determine the interaction dynamics of the bifunctional protein DCoH1, the dimerization cofactor of the transcription factor HNF-1α .

The two disparate functions of DCoH1 (dimerization cofactor of HNF-1)/PCD (pterin-4a-carbinolamine dehydratase) are associated with a change in oligomeric state. DCoH dimers enhance the activity of the diabetes-associated transcription factor HNF-1α (hepatocyte nuclear factor-1α), while the PCD activity of DCoH1 homotetramers aids in aromatic amino acid metabolism. These complexes compete for the same interface of the DCoH dimer. Formation of the DCoH1/HNF-1α complex requires cofolding. The homotetramer of the DCoH1 paralogue, DCoH2, interacts with HNF-1α through simple mixing. To further investigate regulation of DCoH/HNF-1α complex formation, we measured the stability of the DCoH1 homotetramer through unfolding studies by intrinsic tryptophan fluorescence. DCoH2 unfolding is reversible. Surprisingly, the DCoH1 homotetramer is resistant to guanidine unfolding but refolds at a much lower guanidine concentration. We show that a point mutation at the DCoH1 tetramer interface, Thr 51 Ser, overcomes the dissociation barrier of the homotetramer and increases the interaction with HNF-1α. The 1.8 Ǻ resolution crystal structure of DCoH1 T51S shows the presence of an ordered water molecule at the tetramer interface, as in DCoH2, which may destabilize the homotetramer. The equilibrium unfolding data were fit to a two-state model with no apparent intermediate. Folding intermediates were detectable by size exclusion chromatography. For wild-type DCoH1 the intermediates changed with time, suggesting a kinetic origin for the unfolding barrier of the homotetramer. We propose an unfolding pathway in which the tetramer unfolds slowly, but the dimer folds reversibly. Implications for regulation of DCoH1/HNF-1α complex formation are discussed.

Structure of an early intermediate in the M-state phase of the bacteriorhodopsin photocycle.

The structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230 K has been determined by x-ray crystallography to a resolution of 2.0 A. Three-dimensional crystals were trapped by illuminating with actinic light at 230 K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13-C14 double bond appears to be complete, but a relatively large torsion angle of 26 degrees is still seen for the C14-C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (M(N)) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics.

High-resolution structure of the HNF-1alpha dimerization domain.

The N-terminal dimerization domain of the transcriptional activator hepatocyte nuclear factor-1alpha (HNF-1alpha) is essential for DNA binding and association of the transcriptional coactivator, DCoH (dimerization cofactor of HNF-1). To investigate the basis for dimerization of HNF-1 proteins, we determined the 1.2 A resolution X-ray crystal structure of the dimerization domain of HNF-1alpha (HNF-p1). Phasing was facilitated by devising a simple synthesis for Fmoc-selenomethionine and substituting leucine residues with selenomethionine. The HNF-1 dimerization domain forms a unique, four-helix bundle that is preserved with localized conformational shifts in the DCoH complex. In three different crystal forms, HNF-p1 displays subtle shifts in the conformation of the interhelix loop and the crossing angle between the amino- and carboxyl-terminal helices. In all three crystal forms, the HNF-p1 dimers pair through an exposed hydrophobic surface that also forms the binding site for DCoH. Conserved core residues in the dimerization domain of the homologous transcriptional regulator HNF-1beta rationalize the functional heterodimerization of the HNF-1alpha and HNF-1beta proteins. Mutations in HNF-1alpha are associated with maturity-onset diabetes of the young type 3 (MODY3), and the structure of HNF-p1 provides insights into the effects of three MODY3 mutations.

Structural basis of dimerization, coactivator recognition and MODY3 mutations in HNF-1alpha.

Maturity-onset diabetes of the young type 3 (MODY3) results from mutations in the transcriptional activator hepatocyte nuclear factor-1alpha (HNF-1alpha). Several MODY3 mutations target the HNF-1alpha dimerization domain (HNF-p1), which binds the coactivator, dimerization cofactor of HNF-1 (DCoH). To define the mechanism of coactivator recognition and the basis for the MODY3 phenotype, we determined the cocrystal structure of the DCoH-HNF-p1 complex and characterized biochemically the effects of MODY3 mutations in HNF-p1. The DCoH-HNF-p1 complex comprises a dimer of dimers in which HNF-p1 forms a unique four-helix bundle. Through rearrangements of interfacial side chains, a single, bifunctional interface in the DCoH dimer mediates both HNF-1alpha binding and formation of a competing, transcriptionally inactive DCoH homotetramer. Consistent with the structure, MODY3 mutations in HNF-p1 reduce activator function by two distinct mechanisms.

Domain flexibility in retroviral proteases: structural implications for drug resistant mutations.

Rigid body rotation of five domains and movements within their interfacial joints provide a rational context for understanding why HIV protease mutations that arise in drug resistant strains are often spatially removed from the drug or substrate binding sites. Domain motions associated with substrate binding in the retroviral HIV-1 and SIV proteases are identified and characterized. These motions are in addition to closure of the flaps and result from rotations of approximately 6-7 degrees at primarily hydrophobic interfaces. A crystal structure of unliganded SIV protease (incorporating the point mutation Ser 4 His to stabilize the protease against autolysis) was determined to 2.0 A resolution in a new space group, P3221. The structure is in the most "open" conformation of any retroviral protease so far examined, with six residues of the flaps disordered. Comparison of this and unliganded HIV structures, with their respective liganded structures by difference distance matrixes identifies five domains of the protease dimer that move as rigid bodies against one another: one terminal domain encompassing the N- and C-terminal beta sheet of the dimer, two core domains containing the catalytic aspartic acids, and two flap domains. The two core domains rotate toward each other on substrate binding, reshaping the binding pocket. We therefore show that, for enzymes, mutations at interdomain interfaces that favor the unliganded form of the target active site will increase the off-rate of the inhibitor, allowing the substrate greater access for catalysis. This offers a mechanism of resistance to competitive inhibitors, especially when the forward enzymatic reaction rate exceeds the rate of substrate dissociation.

Three-dimensional structures of HIV-1 and SIV protease product complexes.

Strain is eliminated as a factor in hydrolysis of the scissile peptide bond by human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), based on the first eight complexes of products of hydrolysis with the enzymes. The carboxyl group generated at the scissile bond interacts with both catalytic aspartic acids. The structures directly suggest the interactions of the gemdiol intermediate with the active site. Based on the structures, the nucleophilic water is displaced stereospecifically by substrate binding toward one catalytic aspartic acid, while the scissile carbonyl becomes hydrogen bonded to the other catalytic aspartic acid in position for hydrolysis. Crystal structures for two N-terminal (P) products and two C-terminal (Q) products provide unambiguous density for the ligands at 2.2-2.6 A resolution and 17-21% R factors. The N-terminal product, Ac-S-L-N-F/, overlaps closely with the N-terminal sequences of peptidomimetic inhibitors bound to the protease. Comparison of the two C-terminal products, /F-L-E-K and /F(NO2)-E-A-Nle-S, indicates that the P2' residue is highly constrained, while the positioning of the P1' and P3' residues are sequence dependent.

Structure of the protease from simian immunodeficiency virus: complex with an irreversible nonpeptide inhibitor.

A variant of the simian immunodeficiency virus protease (SIV PR), covalently bound to the inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), was crystallized. The structure of the inhibited complex was determined by X-ray crystallography to a resolution of 2.4 A and refined to an R factor of 19%. The variant, SIV PR S4H, was shown to diminish the rate of autolysis by at least 4-fold without affecting enzymatic parameters. The overall root mean square (rms) deviation of the alpha-carbons from the structure of HIV-1PR complexed with a peptidomimetic inhibitor (7HVP) was 1.16 A. The major differences are concentrated in three surface loops with rms differences between 1.2 and 2.1 A. For 60% of the molecule the rms deviation was only 0.6 A. The structure reveals one molecule of EPNP bound per protease dimer, a stoichiometry confirmed by mass spectral analysis. The epoxide moiety forms a covalent bond with either of the active site aspartic acids of the dimer, and the phenyl moiety occupies the P1 binding site. The EPNP nitro group interacts with Arg 8. This structure suggests a starting template for the design of nonpeptide-based irreversible inhibitors of the SIV and related HIV-1 and HIV-2 PRs.

Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae.

Integration of retroviral DNA into the host cell genome requires the interaction of retroviral integrase (IN) protein with the outer ends of both viral long terminal repeats (LTRs) to remove two nucleotides from the 3' ends (3' processing) and to join the 3' ends to newly created 5' ends in target DNA (strand transfer). We have purified the IN protein of human immunodeficiency virus type 1 (HIV-1) after production in Saccharomyces cerevisiae and found it to have many of the properties described for retroviral IN proteins. The protein performs both 3' processing and strand transfer reactions by using HIV-1 or HIV-2 attachment (att) site oligonucleotides. A highly conserved CA dinucleotide adjacent to the 3' processing site of HIV-1 is important for both the 3' processing and strand transfer reactions; however, it is not sufficient for full IN activity, since alteration of nucleotide sequences internal to the HIV-1 U5 CA also impairs IN function, and Moloney murine leukemia virus att site oligonucleotides are poor substrates for HIV-1 IN. When HIV-1 att sequences are positioned internally in an LTR-LTR circle junction substrate, HIV-1 IN fails to cleave the substrate preferentially at positions coinciding with correct 3' processing, implying a requirement for positioning att sites near DNA ends. The 2 bp normally located beyond the 3' CA in linear DNA are not essential for in vitro integration, since mutant oligonucleotides with single-stranded 3' or 5' extensions or with no residues beyond the CA dinucleotide are efficiently used. Selection of target sites is nonrandom when att site oligonucleotides are joined to each other in vitro. We modified an in vitro assay to distinguish oligonucleotides serving as the substrate for 3' processing and as the target for strand transfer. The modified assay demonstrates that nonrandom usage of target sites is dependent on the target oligonucleotide sequence and independent of the oligonucleotide used as the substrate for 3' processing.

Ring opening photoreactions of 5-bromouracil and 5-bromo-2'-deoxyuridine with selected alkylamines.

Several studies in the literature indicate that histones (lysine rich proteins found associated with DNA in eukaryotic chromatin), as well as poly-L-lysine, can be photocross-linked by ultraviolet (UV) light to DNA in which 5-bromo-2'-deoxyuridine has been substituted for thymidine. To gain some insight into the possible nature of this cross-linking, we have studied the photoreactions occurring in deoxygenated aqueous solutions containing 5-bromouracil (I) (BrUra) or 5-bromo-2'-deoxyuridine (III) (BrdUrd) and ethylamine, a lysine side chain analog. In the case of I this reaction produced the ring opened compound N-(N'-ethylcarbamoyl)-3-amino-2-bromoacrylamide (Ia). A small amount of N-(N'-ethylcarbamoyl)-3-ethylamino-2-bromoacrylamide (Ic) was also isolated. It was found that purified Ia, standing in the presence of ethylamine, was gradually converted to Ic in a dark reaction. The beta and alpha anomers of N-(N'-ethylcarbamoyl)-3-(2'deoxyribofuranos-1'-yl) amino-2-bromoacrylamide (IIIa and IIIb respectively) were isolated as products in the photoreaction of III with ethylamine; the alpha anomer was produced in a dark reaction from the beta anomer. The identity of these anomers was established by comparison of their proton NMR spectra with those of the four corresponding alpha and beta furanosyl and pyranosyl isomeric nucleosides of thymine, which are presented in the Appendix. A study was also made of the reaction of I with methylamine; a ring opened product analogous to Ia, viz. N-(N'-methylcarbamoyl)-3-amino-2-bromoacrylamide (IIa) was formed. A similar study with 5-bromo-1-methyluracil produced N-(N'-methylcarbamoyl)-3-methylamino-2-bromoacrylamide (IIc) as a product. Likewise, the reaction of 5-chlorouracil with ethylamine was studied and N-(N'-ethylcarbamoyl)-3-amino-2-chloroacrylamide (Ie), which is analogous in structure to Ia, was found to be produced. Structural identifications were made through use of UV spectroscopy, high resolution 1H-NMR spectroscopy, mass spectrometry and, in the case of Ia and IIa, 13C-NMR spectroscopy. In the BrUra and BrdUrd reaction systems, described above, dehalogenation reactions accounted for a major portion of the products. The yields of ring opened products, determined at pH 10, ranged from a high of 10.3% in the BrUra-ethylamine system to a low of 1.7% in the MeBrUra-methylamine system.(ABSTRACT TRUNCATED AT 400 WORDS)