Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA.

HMG-D is an abundant high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. It is a non-sequence-specific member of a protein family that uses the HMG domain for binding to DNA in the minor groove. The highly charged C-terminal tail of HMG-D contains AK motifs that contribute to high-affinity non-sequence-specific DNA binding. To understand the interactions of the HMG domain and C-terminal tail of HMG-D with DNA in solution, a complex between a high-affinity truncated form of the protein and a disulfide cross-linked DNA fragment was studied using heteronuclear NMR techniques. Despite its relatively high affinity for the single "prebent" site on the DNA, K(d) = 1.4 nM, HMG-D forms a non-sequence-specific complex with the DNA as indicated by exchange broadening of the protein resonances at the DNA interface in solution. The secondary structural elements of the protein are preserved when the protein is complexed with the DNA, and the DNA-binding interface maps to the regions of the protein where the largest chemical shift differences occur. The C-terminal tail of HMG-D confers high-affinity DNA binding, has an undefined structure, and appears to make direct contacts in the major groove of DNA via residues that are potentially regulated by phosphorylation. We conclude that while the HMG domain of HMG-D recognizes DNA with a mode of binding similar to that used by the sequence-specific HMG domain transcription factors, there are noteworthy differences in the structure and interactions of the C-terminal end of the DNA-binding domain and the C-terminal tail.

Co-crystallization and preliminary crystallographic analysis of the high mobility group domain of HMG-D bound to DNA.

Structural studies are essential to understand mechanisms of non-sequence-specific DNA binding used by chromosomal proteins. A non-histone high-mobility group (HMG) chromosomal protein from Drosophila melanogaster, HMG-D, binds duplex DNA in a non-sequence-specific fashion. The DNA-binding domain of HMG-D has been co-crystallized with a duplex DNA fragment in the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell dimensions a = 43.74, b = 53.80, c = 86.84 A. Data have been collected to 2.20 A at 99 K, with diffraction observed to at least 2.0 A. Heavy-atom derivative crystals have been obtained by co-crystallization with oligonucleotides halogenated at major-groove positions near the end of the DNA.

Oxidation of a critical methionine modulates DNA binding of the Drosophila melanogaster high mobility group protein, HMG-D.

HMG-D is a major high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. During overexpression and purification of HMG-D from E. coli, a key DNA binding residue, methionine 13, undergoes oxidation to methionine sulfoxide. Oxidation of this critical residue decreases the affinity of HMG-D for DNA by three-fold, altering the structure of the HMG-D-DNA complex without affecting the structure of the free protein. This work shows that minor modification of DNA intercalating residues may be used to fine tune the DNA binding affinity of HMG domain proteins.

Structural analysis of a novel interaction by calmodulin: high-affinity binding of a peptide in the absence of calcium.

The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of both apocalmodulin and its 1:1 complex with the Neurop peptide have been assigned by triple resonance and nuclear Overhauser effect-(NOE-) based strategies. ApoCaM displays many of the same basic structural features as calcium-saturated calmodulin. Analysis of observed chemical shifts and patterns of NOEs on the main chain indicates extensive and regular secondary structure throughout the N-terminal domain. In contrast, the helices of the C-terminal domain are somewhat irregular and are dynamically averaged. The EF-hands are intact in the N-terminal domain with the loops forming a short antiparallel beta sheet. Under low-salt conditions, two helix-loop-helix EF-hand motifs are present in the C-terminal domain of apoCaM but do not show interstrand NOEs. The spectral perturbations of apoCaM upon complexation with the Neurop peptide are relatively small with the larger chemical shift perturbations occurring in the C-terminal domain. The general secondary structure and tertiary organization appears to remain roughly the same as in free apoCaM. Stoichiometric titration of the apoCaM.Neurop complex with calcium indicates that the C-terminal domain EF-hands have a higher affinity for calcium than N-terminal domain EF-hands. Thus, this complex offers a unique opportunity to examine the structural and energetic consequences of calcium-dependent and calcium-independent binding of peptide to calmodulin.