Characterization of the dimerization domain in the FNR transcription factor

The global anaerobic regulator FNR from Escherichia coli is a dimeric Fe-S protein that is inactivated by O(2) through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activity in vivo. Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization. Because hydrophobic side chains are known to stabilize subunit-subunit interactions between alpha-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an alpha-helix that constitutes a dimerization interface. This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face. In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face. These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O(2) availability.     

Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain

MDC1 is a key mediator of the DNA-damage response in mammals with several phosphorylation-dependent protein interaction domains. The function of its N-terminal forkhead-associated (FHA) domain remains elusive. Here, we show with structural, biochemical and cellular data that the FHA domain mediates phosphorylation-dependent dimerization of MDC1 in response to DNA damage. Crystal structures of the FHA domain reveal a face-to-face dimer with pseudo-dyad symmetry. We found that the FHA domain recognizes phosphothreonine 4 (pT4) at the N-terminus of MDC1 and determined its crystal structure in complex with a pT4 peptide. Biochemical analysis further revealed that in the dimer, the FHA domain binds in trans to pT4 from the other subunit, which greatly stabilizes the otherwise unstable dimer. We show that T4 is phosphorylated primarily by ATM upon DNA damage. MDC1 mutants with the FHA domain deleted or impaired in its ability to dimerize formed fewer foci at DNA-damage sites, but the localization defect was largely rescued by an artificial dimerization module, suggesting that dimerization is the primary function of the MDC1 FHA domain. Our results suggest a novel mechanism for the regulation of MDC1 function through T4 phosphorylation and FHA-mediated dimerization.     

Gypsy and the birth of the SCAN domain

SCAN is a protein domain frequently found at the N termini of proteins encoded by mammalian tandem zinc finger (ZF) genes, whose structure is known to be similar to that of retroviral gag capsid domains and whose multimerization has been proposed as a model for retroviral assembly. We report that the SCAN domain is derived from the C-terminal portion of the gag capsid (CA) protein from the Gmr1-like family of Gypsy/Ty3-like retrotransposons. On the basis of sequence alignments and phylogenetic distributions, we show that the ancestral host SCAN domain (ESCAN for extended SCAN) was exapted from a full-length CA gene from a Gmr1-like retrotransposon at or near the root of the tetrapod animal branch. A truncated variant of ESCAN that corresponds to the annotated SCAN domain arose shortly thereafter and appears to be the only form extant in mammals. The Anolis lizard has a large number of tandem ZF genes with N-terminal ESCAN or SCAN domains. We predict DNA binding sites for all Anolis ESCAN-ZF and SCAN-ZF proteins and demonstrate several highly significant matches to Anolis Gmr1-like sequences, suggesting that at least some of these proteins target retroelements. SCAN is known to mediate protein dimerization, and the CA protein multimerizes to form the core retroviral and retrotransposon capsid structure. We speculate that the SCAN domain originally functioned to target host ZF proteins to retroelement capsids.     

Structural analysis of the transmembrane domain of the epidermal growth factor receptor

The ligand-binding domain of the epidermal growth factor (EGF) receptor is separated from the cytoplasmic protein tyrosine kinase domain by a predicted single transmembrane segment. Antipeptide antibodies prepared against the outer portion of the predicted transmembrane segment confirmed this area was exposed only when cells were treated with permeabilizing agents. To investigate structural requirements for signal transduction by the transmembrane domain, three types of mutant EGF receptor were prepared. The first type was designed to shorten the transmembrane domain, the second to place proline substitutions within this domain, and the third to make amino acid substitutions analogous to those present in the transforming c-erbB2/neu oncoprotein. Mutant human receptors were expressed in null recipient mouse B82L and Chinese hamster ovary cells. All receptors bound EGF and exhibited EGF-stimulated protein tyrosine kinase activity in vivo as assayed using a 125I-labeled monoclonal anti-phosphotyrosine antibody. EGF stimulated growth of cells expressing each mutant receptor with similar dose-response characteristics. In contrast to other growth factor receptors, the transmembrane domain of the EGF receptor is tolerant to a variety of changes which neither mimic EGF action by constitutive activation nor interfere with ligand-induced signal transduction.     

Structural characterization of a dimerization interface in the CD28 transmembrane domain

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Structural features of the KPI domain control APP dimerization, trafficking, and processing

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.     

Structural basis for GTP-dependent dimerization of hydrogenase maturation factor HypB

Maturation of [NiFe]-hydrogenase requires the insertion of iron, cyanide and carbon monoxide, followed by nickel, to the catalytic core of the enzyme. Hydrogenase maturation factor HypB is a metal-binding GTPase that is essential for the nickel delivery to the hydrogenase. Here we report the crystal structure of Archeoglobus fulgidus HypB (AfHypB) in apo-form. We showed that AfHypB recognizes guanine nucleotide using Asp-194 on the G5 loop despite having a non-canonical NKxA G4-motif. Structural comparison with the GTPγS-bound Methanocaldococcus jannaschii HypB identifies conformational changes in the switch I region, which bring an invariant Asp-72 to form an intermolecular salt-bridge with another invariant residue Lys-148 upon GTP binding. Substitution of K148A abolished GTP-dependent dimerization of AfHypB, but had no significant effect on the guanine nucleotide binding and on the intrinsic GTPase activity. In vivo complementation study in Escherichia coli showed that the invariant lysine residue is required for in vivo maturation of hydrogenase. Taken together, our results suggest that GTP-dependent dimerization of HypB is essential for hydrogenase maturation. It is likely that a nickel ion is loaded to an extra metal binding site at the dimeric interface of GTP-bound HypB and transferred to the hydrogenase upon GTP hydrolysis.     

Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces

S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G. Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy. The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core. Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing. Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K(D) value in the millimolar range. The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E. Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K(D)=160(+/-40)microM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains. This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain. Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al. in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme.     

ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate

The nuclear zinc finger protein ZFPIP/Zfp462 is an important factor involved in cell division during the early embryonic development of vertebrates. In pluripotent P19 cells, ZFPIP/Zfp462 takes part in cell proliferation, likely via its role in maintaining chromatin structure. To further define the function of ZFPIP/Zfp462 in the mechanisms of pluripotency and cell differentiation, we constructed a stable P19 cell line in which ZFPIP/Zfp462 knockdown is inducible.We report that ZFPIP/Zfp462 was vital for mitosis and self-renewal in pluripotent P19 cells. Its depletion induced substantial decreases in the expression of the pluripotency genes Nanog, Oct4 and Sox2 and was associated with the transient expression of specific neuronal differentiation markers. We also demonstrated that ZFPIP/Zfp462 expression appears to be unnecessary after neuronal differentiation is induced in P19 cells.Taken together, our results strongly suggest that ZFPIP/Zfp462 is a key chromatin factor involved in maintaining P19 pluripotency and in the early mechanisms of neural differentiation but that it is dispensable in differentiated P19 cells.     

Characterization of an amino-terminal dimerization domain from retroviral restriction factor Fv1

The Fv1 protein is an endogenous factor in mice that confers resistance to infection by certain classes of murine leukemia virus, a phenomenon referred to as restriction. The mechanism of restriction is not understood, and the low endogenous level of Fv1 in cells has prevented any biochemical or biophysical analysis of the protein. We have now purified recombinant Fv1(n) protein from a baculovirus system and demonstrate that Fv1 exists in a multimeric form. Furthermore, we have mapped the position of two domains within the protein using limited proteolysis. Biophysical characterization of the N-terminal domain reveals that it comprises a highly helical and extended dimeric structure. Based on these biochemical and biophysical data, we propose a model for the arrangement of domains in Fv1 and suggest that dimerization of the N-terminal domain is necessary for Fv1 function to allow the protein to interact with multiple capsid protomers in retroviral cores.