Markus Zeeb

Common mode of DNA binding to cold shock domains Crystal structure of hexathymidine bound to the domain-swapped form of a major cold shock protein from Bacillus caldolyticus

Bacterial cold shock proteins (CSPs) regulate cellular adaptation to cold stress. Functions ascribed to CSP include roles as RNA chaperones and in transcription antitermination. We present the crystal structure of the Bacillus caldolyticus CSP (Bc‐Csp) in complex with hexathymidine (dT6) at a resolution of 1.29 Å. Bound to dT6, crystalline Bc‐Csp forms a domain‐swapped dimer in which β strands 1–3 associate with strands 4 and 5 from the other subunit to form a closed β barrel and vice versa. The globular units of dimeric Bc‐Csp closely resemble the well‐known structure of monomeric CSP. Structural reorganization from the monomer to the domain‐swapped dimer involves a strictly localized change in the peptide bond linking Glu36 and Gly37 of Bc‐Csp. Similar structural reorganizations have not been found in any other CSP or oligonucleotide/oligosaccharide‐binding fold structures. Each dT6 ligand is bound to one globular unit of Bc‐Csp via an amphipathic protein surface. Individual binding subsites interact with the DNA bases through stacking and hydrogen bonding. The sugar–phosphate backbone remains solvent exposed. Based on crystallographic and biochemical studies of deoxyoligonucleotide binding to CSP, we suggest a common mode of binding of single‐stranded heptanucleotide motifs to proteins containing cold shock domains, including the eukaryotic Y‐box factors.

Single-stranded DNA binding of the cold-shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization

Cold‐shock proteins (CSPs) bind to single‐stranded nucleic acids, thereby acting as a “RNA chaperone.” To gain deeper insights into the rather unspecific nature of ssDNA/RNA binding, we characterized the binding interface of CspB from Bacillus subtilis to a 25‐mer of ssDNA (Y‐Box25) using heteronuclear 2D NMR spectroscopy. Seventeen residues, including eight out of nine aromatic amino acids, are directly involved in the Y‐Box25 interaction and were identified by extreme line broadening of their cross‐peaks. Eight residues belong to the earlier proposed RNP binding motifs. A second set of seven backbone amides becomes evident by major chemical shift perturbations reporting remote conformational rearrangements upon binding. These residues are located in loop β3–β4 and loopβ4–β5, and include Ile18. The individual contributions of the so‐identified residues were examined by fluorescence titration experiments of 15 CspB variants. Phenylalanine substitutions in‐ and outside the RNP motifs significantly reduce the binding affinity. Unrestricted possible backbone conformations of loop β3–β4 also markedly contribute to binding. Stopped‐flow fluorescence kinetics revealed that the different binding affinities of CspB variants are determined by the dissociation rate, whereas the association rate remains unchanged. This might be of importance for the “RNA chaperone” activity of CspB.

Recognition of T-rich single-stranded DNA by the cold shock protein Bs-CspB in solution

Cold shock proteins (CSP) belong to the family of single-stranded nucleic acid binding proteins with OB-fold. CSP are believed to function as ‘RNA chaperones’ and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT7) by NMR spectroscopy. Bs-CspB reveals an almost invariant conformation when bound to dT7 with only minor reorientations in loop β1–β2 and β3–β4 and of few aromatic side chains involved in base stacking. Binding studies of protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. 15N NMR relaxation and H/D exchange experiments revealed that binding of dT7 increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire protein and especially of loop β3–β4.

Single-stranded DNA bound to bacterial cold-shock proteins: Preliminary crystallographic and Raman analysis

The cold-shock response has been described for several bacterial species. It is characterized by distinct changes in intracellular protein patterns whereby a set of cold-shock-inducible proteins become abundant. The major cold-shock proteins of Bacillus subtilis (Bs-CspB) and Bacillus caldolyticus (Bc-Csp) are small oligonucleotide/oligosaccharide-binding (OB) fold proteins that have been described as binding single-stranded nucleic acids. Bs-CspB (Mr = 7365) and Bc-Csp (Mr = 7333) were crystallized in the presence of the deoxyhexanucleotide (dT)6. Crystals of (dT)6 with Bs-CspB grew in the orthorhombic space group C222(1), with unit-cell parameters a = 49.0, b = 53.2, c = 77.0 A. Crystals with Bc-Csp grew in the primitive orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 74.3, b = 64.9, c = 31.2 A. These crystals diffract to maximal resolutions of 1.78 and 1.29 A, respectively. The presence of protein and DNA in the crystals was demonstrated by Raman spectroscopy.

Structural Basis for Interaction of the Tandem Zinc Finger Domains of Human Muscleblind with Cognate RNA from Human Cardiac Troponin T

The human muscleblind-like proteins (MBNL) regulate tissue-specific splicing by targeting cardiac troponin T and other pre-mRNAs; aberrant targeting of CUG and CCUG repeat expansions frequently accompanies the neuromuscular disease myotonic dystrophy. We show, using biolayer interferometry (Octet) and NMR spectroscopy, that the zinc finger domains of MBNL isoform 1 (MBNL1) are necessary and sufficient for binding CGCU sequences within the pre-mRNA of human cardiac troponin T. Protein constructs containing zinc fingers 1 and 2 (zf12) and zinc fingers 3 and 4 (zf34) of MBNL1 each fold into a compact globular tandem zinc finger structure that participates in RNA binding. NMR spectra show that the stoichiometry of the interaction between zf12 or zf34 and the CGCU sequence is 1:1, and that the RNA is single-stranded in the complex. The individual zinc fingers within zf12 or zf34 are nonequivalent: the primary RNA binding surface is formed in each pair by the second zinc finger (zf2 or zf4), which interacts with the CGCU RNA sequence. The NMR structure of the complex between zf12 and a 15-base RNA of sequence 95GUCUCGCUUUUCCCC109, containing a single CGCU element, shows the single-stranded RNA wrapped around zf2 and extending to bind to the C-terminal helix. Bases C101, U102, and U103 make well-defined and highly ordered contacts with the protein, whereas neighboring bases are less well-ordered in the complex. Binding of the MBNL zinc fingers to cardiac troponin T pre-mRNA is specific and relatively simple, unlike the complex multiple dimer–trimer stoichiometries postulated in some previous studies.

Design and characterization of a soluble fragment of the extracellular ligand-binding domain of the peptide hormone receptor guanylyl cyclase-C

The intestinal guanylyl cyclase-C (GC-C) was originally identified as an Escherichia coli heat-stable enterotoxin (STa) receptor. STa stimulates GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin, causing severe diarrhea. To investigate the interactions of the endogenous and bacterial ligands with GC-C, we designed and characterized a soluble and properly folded fragment of the extracellular ligand-binding domain of GC-C. The membrane-bound guanylyl cyclases exhibit a single transmembrane spanning helix and a globularly folded extracellular ligand-binding domain that comprises about 410 of 1050 residues. Based on the crystal structure of the dimerized-binding domain of the guanylyl cyclase-coupled atrial natriuretic peptide receptor and a secondary structure-guided sequence alignment, we generated a model of the extracellular domain of GC-C comprised of two subdomains. Mapping of mutational and cross-link data onto this structural model restricts the ligand-binding region to the membrane proximal subdomain. We thus designed miniGC-C, a 197 amino acid fragment that mimics the ligand-binding membrane proximal subdomain. Cloning, expression and spectroscopic studies reveal miniGC-C to be a soluble and properly folded protein with a distinct secondary and tertiary structure. MiniGC-C binds STa with nanomolar affinity.

Millisecond Protein Folding Studied by NMR Spectroscopy

Proteins are involved in virtually every biological process and in order to function, it is necessary for these polypeptide chains to fold into the unique, native conformation. This folding process can take place rapidly. NMR line shape analyses and transverse relaxation measurements allow protein folding studies on a microsecond-to-millisecond time scale. Together with an overview of current achievements within this field, we present millisecond protein folding studies by NMR of the cold shock protein CspB from Bacillus subtilis.

Structure-based stability analysis of an extremely stable dimeric DNA binding protein from Sulfolobus islandicus.

ORF56 is a small and thermodynamically extremely stable dimeric protein from the archaeon Sulfolobus islandicus. This DNA binding protein is encoded on plasmid pRN1 and possibly controls the copy number of the plasmid. We report the solution NMR structure as well as the crystal structure of ORF56 comprising a ribbon-helix-helix fold. The homodimer consists of an antiparallel intersubunit beta-sheet and two alpha-helices per monomer, which is a common DNA binding fold of plasmid- and phage-encoded gene regulation proteins. NMR titration experiments with ORF56 and double-stranded DNA derived from its promoter binding site revealed that it is largely the beta-sheets that interact with the DNA. The beta-sheet experiences high local fluctuations, which are conserved among DNA binding ribbon-helix-helix dimers from mesophilic and hyperthermophilic organisms. In contrast, residues strongly protected against H-D exchange are localized in helix 2, forming the hydrophobic intermolecular core of the dimer. A structure-based comparison of the intermolecular binding surface and the change in accessible surface area upon unfolding of various ribbon-helix-helix dimers with the Gibbs free energy changes and m values show a correlation between hydrophobicity of these surface areas and stability. These findings provide possible explanations for the very high thermodynamic stability of ORF56 with retained DNA binding capacity.