摘要：

The structure of bovine pancreatic deoxyribonuclease I (DNase I) has been refined at 2 resolution using the restrained parameter, reciprocal least-squares procedure of Hendrickson and Konnert. The conventional R-factor for 16,104 reflections with I ≥ 3σ( I) from 6.0 to 2.0 resolution is 0.157. Bond lengths and angles of the refined structure are close to ideal values with root-mean-square (r.m.s.) deviations of 0.023 and 1.4 °, respectively. The r.m.s. deviation of short non-bonded contacts from the sum of van der Waals' radii is 0.18 . The orientation of side-chains shows a clear trimodal distribution of χ 1-angles at 60 °, 180 °, 60 ° (in the order of preference) corresponding to staggered conformations. The chemically determined sequence was corrected at four positions, the major correction being an insertion of the tripeptide Ile-Val-Arg between Arg27 and Arg28. Extended hydrophobic regions in between, and on either side of, the two central six-stranded β-pleated sheets are mainly responsible for the low average isotropic temperature factor of 11.9 2 for the 2033 protein atoms. Besides the flexible loop region between Gly97 and Gly102 (Glu99 and Ser100 are disordered) and the carbohydrate side-chain, which both extend into a large solvent channel, only the exposed loop Arg70 to Lys74 shows elevated thermal mobility. The longest of the eight helices in DNase I, together representing 26% of the structure, has a 22 ° kink and consists of two α-helical segments (residues 136 to 144 and 145 to 155) separated by a 3 10-helical turn. DNase I fragments 1 to 120 and 121 to 257 can be superimposed by an approximate 2-fold axis (r.m.s. deviation 1.49 for 61 equivalent C α positions), suggesting that the enzyme might be the result of gene duplication. The two Ca 2+ bound to DNase I under crystallization conditions are important for its structural integrity by stabilizing the surface loop Asp198 to Thr204 and limiting the region of high thermal mobility in the flexible loop to residues Gly97 to Gly102. The N-linked carbohydrate side-chain attached to Asn 18 is of the high-mannose type with a branching point at the mannose residue in position 3. Two N-acetylglucosamine residues, which are in contact with the protein, and the following mannose are well defined and show the usual β-1,4-linkage in an extended conformation, whereas the positions of four further mannose residues are less reliable due to increasingly higher thermal motion and possibly microheterogeneity. A total of 377 crystallographically independent solvent sites has been determined in difference Fourier maps and their positions and temperature factors have been refined ( 〈B〉 = 30 A 2). A possible mechanism of action and a model for the interaction with double-stranded DNA involving the binding of an exposed loop (Arg70 to Lys74) in the minor groove is discussed.

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