The structure determined for the HMG-box motif from HMG-D is essentially identical to the structure determined for the B-domain of mammalian HMG-1. Since these proteins have significantly different sequences our results indicate that the global fold and the mode of interaction with DNA are also likely to be conserved in all eukaryotes.

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The backbone dynamics of uniformly 15N-labeled chymotrypsin inhibitor 2 (CI2) and of the complex formed by the association of two fragments consisting of residues 20-59 and 60-83 have been studied. A data set consisting of 15N longitudinal (T1) and transverse (T1 rho) relaxation times and (1H)-15N NOE enhancements has been measured for all backbone NH groups in both proteins. Information on internal motions has been extracted from these data using the model-free approach to determine order parameters (S2) and effective internal correlation times (tau e). The data indicate that most of the backbone of CI2 is highly constrained (S2 approximately 0.9) with the exception of residues in the binding loop (residues 54-64), which have slightly lower order parameters. Most of the residues in the CI2(20-59).(60-83) complex are also highly constrained (S2 approximately 0.9). However, the loss of the covalent bond between Met59 and Glu60 leads to a large increase in the mobility of residues in the loop region. The residues in the first half of the loop region have significantly lower order parameters than those in the second half of the loop. This observation suggests that the NH2 group that is released on cleavage of the scissile bond remains anchored in its original position, inhibiting the attack of water on the acyl-enzyme that is formed between the protease and the cleaved inhibitor. More importantly, the NH2 group is optimally placed for reversing the formation of the acyl-enzyme so that the equilibrium between the cleaved and uncleaved inhibitor, bound to the protease, greatly favors the uncleaved complex.

Comparison of the Fyn SH2 domain structure with other structures of SH2 domains highlights several interesting features. Conservation of helix capping interactions among various SH2 domains is suggestive of a role in protein stabilisation. The presence of a type-I' turn in the BG loop, which is dependent on the presence of a glycine residue at position BG3, is indicative of a binding pocket, characteristic of the Src family, SykC and Abl, rather than a binding groove found in PLC-gamma 1C, p85 alpha N and Shc, for example.