Structure of free BglII reveals an unprecedented scissor-like motion for opening an endonuclease.

Restriction endonuclease BglII completely encircles its target DNA, making contacts to both the major and minor grooves. To allow the DNA to enter and leave the binding cleft, the enzyme dimer has to rearrange. To understand how this occurs, we have solved the structure of the free enzyme at 2.3 A resolution, as a complement to our earlier work on the BglII-DNA complex. Unexpectedly, the enzyme opens by a dramatic 'scissor-like' motion, accompanied by a complete rearrangement of the alpha-helices at the dimer interface. Moreover, within each monomer, a set of residues--a 'lever'--lowers or raises to alternately sequester or expose the active site residues. Such an extreme difference in free versus complexed structures has not been reported for other restriction endonucleases. This elegant mechanism for capturing DNA may extend to other enzymes that encircle DNA.

BglII and MunI: what a difference a base makes.

Restriction endonucleases are resilient to alterations in their DNA-binding specificities. Structures of the BglII and MunI endonucleases bound to their palindromic DNA sites, which differ by only their outer base pairs from the recognition sequences of BamHI and EcoRI, respectively, have recently been determined. A comparison of these complexes reveals surprising differences and similarities in structure, and provides a basis for understanding the immutability of restriction endonucleases.

Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution.

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1. 5 A resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar alpha/beta core, but in BglII, the core is augmented by a beta-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein-DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases.

Alzheimer's peptide Abeta1-42 binds to two beta-sheets of alpha1-antichymotrypsin and transforms it from inhibitor to substrate.

The serpin alpha1-antichymotrypsin is a major component of brain amyloid plaques in Alzheimer's disease. In vitro alpha1-antichymotrypsin interacts with the Alzheimer's amyloid peptide Abeta1-42 and stimulates both formation and disruption of neurotoxic Abeta1-42 fibrils in a concentration-dependent manner. We have constructed a new hybrid model of the complex between Abeta1-42 and alpha1-antichymotrypsin in which both amino and carboxyl sequences of Abeta1-42 insert into two different beta-sheets of alpha1-antichymotrypsin. We have tested this model and shown experimentally that full-length and amino-terminal segments of Abeta1-42 bind to alpha1-antichymotrypsin as predicted. We also show that Abeta1-42 forms both intra- and intermolecular SDS-stable complexes with alpha1-antichymotrypsin and that the binding of Abeta1-42 to alpha1-antichymotrypsin abolishes the inhibitory activity of the latter and its ability to form stable complex with chymotrypsin. The existence of both inter- as well as intramolecular complexes of Abeta1-42 explains the nonlinear concentration-dependent effects of alpha1-antichymotrypsin on Abeta1-42 fibril formation, which we have reinvestigated here over a broad range of Abeta1-42:alpha1-antichymotrypsin ratios. These data suggest a molecular basis for the distinction between amorphous and fibrillar Abeta1-42 in vivo. The reciprocal effects of Abeta1-42 and alpha1-antichymotrypsin could play a role in the etiology of Alzheimer's disease.

Engineering an anion-binding cavity in antichymotrypsin modulates the "spring-loaded" serpin-protease interaction.

Expressed in a kinetically trapped folding state, a serpin couples the thermodynamic driving force of a massive beta-sheet rearrangement to the inhibition of a target protease. Hence, the serpin-protease interaction is the premier example of a "spring-loaded" protein-protein interaction. Amino acid substitutions in the hinge region of a serpin reactive loop can weaken the molecular spring, which converts the serpin from an inhibitor into a substrate. To probe the molecular basis of this conversion, we report the crystal structure of A349R antichymotrypsin in the reactive loop cleaved state at 2.1 A resolution. This amino acid substitution does not block the beta-sheet rearrangement despite the burial of R349 in the hydrophobic core of the cleaved serpin along with a salt-linked acetate ion. The inhibitory activity of this serpin variant is not obliterated; remarkably, its inhibitory properties are anion-dependent due to the creation of an anion-binding cavity in the cleaved serpin.

Arginine substitutions in the hinge region of antichymotrypsin affect serpin beta-sheet rearrangement.

A hallmark of serpin function is the massive beta-sheet rearrangement involving the insertion of the cleaved reactive loop into beta-sheet A as strand s4A. This structural transition is required for inhibitory activity. Small hydrophobic residues at P14 and P12 positions of the reactive loop facilitate this transition, since these residues must pack in the hydrophobic core of the cleaved serpin. Despite the radical substitution of arginine at the P12 position, the crystal structure of cleaved A347R antichymotrypsin reveals full strand s4A insertion with normal beta-sheet A geometry; the R347 side chain is buried in the hydrophobic protein core. In contrast, the structure of cleaved P14 T345R antichymotrypsin reveals substantial yet incomplete strand s4A insertion, without burial of the R345 side chain.

Is the binding of beta-amyloid protein to antichymotrypsin in Alzheimer plaques mediated by a beta-strand insertion?

A growing body of experimental evidence demonstrates that the serpin antichymotrypsin plays a regulatory role in Alzheimer plaque physiology by interacting with the 42 residue beta-amyloid protein, and we have used molecular modeling and energy minimization techniques to study this interaction. Based on the unique plasticity of beta-sheet elements in antichymotrypsin (as well as other serpins), we conclude that the interaction of the two proteins is mediated by insertion of the N-terminus of beta-amyloid into beta-sheet C of antichymotrypsin as a pseudo-strand s1C. This beta-strand insertion requires the displacement of native antichymotrypsin strand s1C, which is known to occur partially or completely at different stages of serpin function. Thus, the association of the two proteins in vivo may be facilitated by a particular functional state of the serpin, e.g., the native or protease-complexed state.

Structural change in alpha-chymotrypsin induced by complexation with alpha 1-antichymotrypsin as seen by enhanced sensitivity to proteolysis.

Both human neutrophil elastase (HNE) and free chymotrypsin (Chtr) proteolyze Chtr within the complex that Chtr forms with antichymotrypsin (ACT). As free Chtr is stable both to self-digestion and to digestion by HNE, these results are indicative of a stability and/or conformational change in Chtr that accompanies complex formation. As determined by both N-terminal sequence analysis and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS), the major initial sites of HNE cleavage of complexed Chtr are between gamma-chain residues A158/S159 and V188/S189. Significantly, this latter site is at the base of the S1 site that recognizes the P1 position of the serpin. A slower cleavage in the beta-chain between T139/G140 is also found. In addition, rACT is cleaved between residues V22/D23. The gamma-chain of complexed Chtr is also cleaved by free Chtr, but at different sites: L162/L163 and W172/G173. beta-Chain cleavages were also found between residues Q81/K82 and F114/S115. Cleavages similar to those described above were also found when Chtr was complexed with the L358F-rACT variant, but not for Chtr complexed with either of the smaller inhibitors bovine pancreatic trypsin inhibitor or turkey ovomucoid third domain, nor for the covalent adduct of Chtr with N-p-tosylphenylalanyl chloromethyl ketone. We conclude that the structural change in Chtr making it a proteinase substrate is coupled with the large conformational change in ACT following complex formation. Complexed Chtr is much less reactive toward proteolytic digestion in the presence of high salt than in its absence, in accord with the high-salt induced release of active enzyme from the Chtr.rACT complex and the suggestion that electrostatic interactions mediate the coupling of structural change between rACT and Chtr within the Chtr.rACT complex. Potential physiological consequences of this work are explored.