Backbone Dynamics of the A-Domain of HMG1 As Studied by 15N NMR Spectroscopy

Abstract

The HMG-box sequence motif (approximately 80 residues) occurs in a number of abundant eukaryotic chromosomal proteins such as HMG1, which binds DNA without sequence specificity, but with "structure specificity", as well as in several sequence-specific transcription factors. HMG1 has two such boxes, A and B, which show approximately 30% sequence identity, and an acidic C-terminal tail. The boxes are responsible for the ability of the protein to bend DNA and bind to bent or distorted DNA. The structure of the HMG box has been determined by NMR spectroscopy for the B-domain of HMG1 [Weir et al. (1993) EMBO J. 12, 1311-1319; Read et al. (1993) Nucleic Acids Res. 21, 3427-3436) and for Drosophila HMG-D (Jones et al. (1994) Structure 2, 609-627]. It has an unusual twisted L-shape, suggesting that the protein might tumble anisotropically in solution. In this paper we report studies of the A-domain from HMG1 using 15N NMR spectroscopy which show that the backbone dynamics of the protein can be described by two different rotational correlation times of 9.0 +/- 0.5 and 10.8 +/- 0.5 ns. We show that the relaxation data can be analyzed by assuming that the protein is a rigid, axially symmetric ellipsoid undergoing anisotropic rotational diffusion; the global rotational diffusion constants, D parallel and D perpendicular, were estimated as 2.47 x 10(7) and 1.49 x 10(7) s-1, respectively. By estimating the angle between the amide bond vectors and the major axis of the rotational diffusion tensor from the family of structures determined by NMR spectroscopy [see accompanying paper, Hardman et al. (1995) Biochemistry 34, 16596-16607], we were able to show that the ellipsoid spectral density equation can reproduce the major features of the 15N T1 and T2 profiles of the three helices in the HMG1 A-domain. The backbone dynamics of the A-domain were then compared with those of the B-domain and the HMG box from HMG-D. This comparison strongly supported the differences observed in the orientation of helix I in the three structures, where the B-domain appears to be more similar to HMG-D than it is to the A-domain. These differences may turn out to be related to subtle differences in the DNA-binding properties of the A- and B-domains of HMG1.

Tail-Mediated Collapse of HMGB1 Is Dynamic and Occurs via Differential Binding of the Acidic Tail to the A and B Domains

“…42,46 The A and B domains were found to have a rotational correlation time of ∼ 10 ns and, as expected from the twisted L shape, to tumble anisotropically in solution. The anisotropy is strongly apparent from the raw data, in which the residues in helices fall into two groups.…”

Structure of a Complex of Tandem HMG Boxes and DNA

“…Although the A and B boxes possess the same global fold, they differ in detail, predominantly in the orientation of helices I and II with respect to the rest of the molecule, and in the length of the loop between these helices, which is longer in the A box. 1,14 The structures of the HMG boxes of HMG-D, 15 Sox-4 16 and Nhp6a 17 have been determined by NMR spectroscopy and all are very similar to the structures of the A and B boxes from HMGB1, resembling most closely the B box. 1 Structures of single HMG boxes bound to DNA have been determined for a number of proteins, both sequence-specific and non-sequence-specific.…”

Contributions to protein entropy and heat capacity from bond vector motions measured by NMR spin relaxation

“…Equation (7) is strictly valid in the case of a clear separation of the time-scales for overall and internal motions and for isotropic motion. Not all molecules tumble isotropically, however, and a number of methods have been described in the literature for obtaining dynamical information on molecules that diffuse anisotropically in solution (Broadhurst et al, 1995;Bruschweiler et al, 1995;Tjandra et al, 1995;Zheng et al, 1995). In principle, for a rigid molecule with axial symmetry three correlation times are required to describe rotational diffusion, while in the general case ®ve correlation times are necessary for a rigid ellipsoid (Woessner, 1962).…”