NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation.

We describe the NMR structure of DsbB, a polytopic helical membrane protein. DsbB, a bacterial cytoplasmic membrane protein, plays a key role in disulfide bond formation. It reoxidizes DsbA, the periplasmic protein disulfide oxidant, using the oxidizing power of membrane-embedded quinones. We determined the structure of an interloop disulfide bond form of DsbB, an intermediate in catalysis. Analysis of the structure and interactions with substrates DsbA and quinone reveals functionally relevant changes induced by these substrates. Analysis of the structure, dynamics measurements, and NMR chemical shifts around the interloop disulfide bond suggest how electron movement from DsbA to quinone through DsbB is regulated and facilitated. Our results demonstrate the extraordinary utility of NMR for functional characterization of polytopic integral membrane proteins and provide insights into the mechanism of DsbB catalysis.

The tetramer structure of the Nervy homology two domain, NHR2, is critical for AML1/ETO's activity.

AML1/ETO is the chimeric protein resulting from the t(8;21) in acute myeloid leukemia. The Nervy homology 2 (NHR2) domain in ETO mediates oligomerization and AML1/ETO's interactions with ETO, MTGR1, and MTG16, and with the corepressor molecules mSin3A and HDAC1 and HDAC3. We solved the NHR2 domain structure and found it to be an alpha-helical tetramer. We show that oligomerization contributes to AML1/ETO's inhibition of granulocyte differentiation, is essential for its ability to enhance the clonogenic potential of primary mouse bone marrow cells, and affects AML1/ETO's activity on several endogenous genes. Oligomerization is also required for AML1/ETO's interactions with ETO, MTGR1, and MTG16, but not with other corepressor molecules.

Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.

The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33-239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33-239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33-239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33-239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33-239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33-239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33-239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle.

CBFbeta allosterically regulates the Runx1 Runt domain via a dynamic conformational equilibrium.

Core binding factors (CBFs) are heterodimeric transcription factors consisting of a DNA-binding CBFalpha subunit and non-DNA-binding CBFbeta subunit. The CBFbeta subunit increases the affinity of the DNA-binding Runt domain of CBFalpha for DNA while making no direct contacts to the DNA. We present evidence for conformational exchange in the S-switch region in a Runt domain-DNA complex that is quenched upon CBFbeta binding. Analysis of (15)N backbone relaxation parameters shows that binding of CBFbeta reduces the backbone dynamics in the microsecond-to-millisecond time frame for several regions of the Runt domain that make energetically important contacts with the DNA. The DNA also undergoes conformational exchange in the Runt domain-DNA complex that is quenched in the presence of CBFbeta. Our results indicate that allosteric regulation by the CBFbeta subunit is mediated by a shift in an existing dynamic conformational equilibrium of both the Runt domain and DNA.

Molecular basis for substrate-dependent transmembrane signaling in an outer-membrane transporter.

Transmembrane signaling events that propagate through receptors and transporters have critical roles in cellular function and regulation. In the Escherichia coli vitamin B(12) transporter, BtuB, substrate binding to the extracellular surface of the protein triggers the unfolding of an energy coupling motif at the periplasmic surface. Here, the molecular interactions mediating this substrate-dependent transmembrane signaling event were investigated in a novel way by combining a two mutant cycle analysis with site-directed spin labeling (SDSL). SDSL was used to monitor the unfolding and conformational equilibrium of the energy-coupling motif, and a thermodynamic two-mutant cycle analysis was used to estimate pair-wise interaction free energies for a pair of charged residues (D316 and R14) within the protein interior. The data indicate that D316 and R14 are critical to this structural transition. Substrate binding is shown to reduce the interaction free energy between these residues, thereby triggering the unfolding of the energy coupling motif of this membrane transporter. The result indicates that SDSL when used in combination with a mutant cycle analysis provides an approach to examine the molecular interactions mediating signaling events in membrane proteins.

High resolution structure of the HDGF PWWP domain: a potential DNA binding domain.

Hepatoma Derived Growth Factor (HDGF) is an endogenous nuclear-targeted mitogen that is linked with human disease. HDGF is a member of the weakly conserved PWWP domain family. This 70-amino acid motif, originally identified from the WHSC1 gene, has been found in more than 60 eukaryotic proteins. In addition to the PWWP domain, many proteins in this class contain known chromatin remodeling domains, suggesting a role for HDGF in chromatin remodeling. We have determined the NMR structure of the HDGF PWWP domain to high resolution using a combination of NOEs, J-couplings, and dipolar couplings. Comparison of this structure to a previously determined structure of the HDGF PWWP domain shows a significant difference in the C-terminal region. Comparison to structures of other PWWP domains shows a high degree of similarity to the PWWP domain structures from Dnmt3b and mHRP. The results of selected and amplified binding assay and NMR titrations with DNA suggest that the HDGF PWWP domain may function as a nonspecific DNA-binding domain. Based on the NMR titrations, we propose a model of the interaction of the PWWP domain with DNA.

Structure of the MLL CXXC domain-DNA complex and its functional role in MLL-AF9 leukemia.

The gene MLL (encoding the protein mixed-lineage leukemia) is the target of chromosomal translocations that cause leukemias with poor prognosis. All leukemogenic MLL fusion proteins retain the CXXC domain, which binds to nonmethylated CpG DNA sites. We present the solution structure of the MLL CXXC domain in complex with DNA, showing how the CXXC domain distinguishes nonmethylated from methylated CpG DNA. On the basis of the structure, we generated point mutations that disrupt DNA binding. Introduction of these mutations into the MLL-AF9 fusion protein resulted in increased DNA methylation of specific CpG nucleotides in Hoxa9, increased H3K9 methylation, decreased expression of Hoxa9-locus transcripts, loss of immortalization potential, and inability to induce leukemia in mice. These results establish that DNA binding by the CXXC domain and protection against DNA methylation is essential for MLL fusion leukemia. They also provide support for viewing this interaction as a potential target for therapeutic intervention.

Structural and functional characterization of Runx1, CBF beta, and CBF beta-SMMHC.

Core binding factors (CBFs) are heterodimeric transcription factors consisting of a DNA-binding CBF alpha subunit and non-DNA-binding CBF beta subunit. DNA binding and heterodimerization is mediated by a single domain in the CBF alpha subunit called the Runt domain, while sequences flanking the Runt domain confer other biochemical activities such as transactivation. On the other hand, the heterodimerization domain in CBF beta is the only functional domain that has been identified in this subunit. The biophysical properties of the Runt domain and the CBF beta heterodimerization domain, and the structures of the isolated domains as well as of the Runt domain-DNA, Runt domain-CBF beta, and ternary Runt domain-CBF beta-DNA complexes, have been characterized over the past several years, and are summarized in this review.

Altered affinity of CBF beta-SMMHC for Runx1 explains its role in leukemogenesis.

Chromosomal translocations involving the human CBFB gene, which codes for the non-DNA binding subunit of CBF (CBF beta), are associated with a large percentage of human leukemias. The translocation inv(16) that disrupts the CBFB gene produces a chimeric protein composed of the heterodimerization domain of CBF beta fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (CBF beta-SMMHC). Isothermal titration calorimetry results show that this fusion protein binds the Runt domain from Runx1 (CBF alpha) with higher affinity than the native CBF beta protein. NMR studies identify interactions in the CBF beta portion of the molecule, as well as the SMMHC coiled-coil domain. This higher affinity provides an explanation for the dominant negative phenotype associated with a knock-in of the CBFB-MYH11 gene and also helps to provide a rationale for the leukemia-associated dysregulation of hematopoietic development that this protein causes.