Journal:Acta Cryst D:S0907444911047251
Flexibility of the flap in the active site of BACE1 as revealed by crystal structures and molecular dynamics simulationsYechun Xu, Minjun Li, Harry Greenblatt, Wuyan Chen, Aviv Paz, Orly Dym, Yoav Peleg, Tiantian Chen, Xu Shen, Jianhua He, Hualiang Jiang, Israel Silman & Joel L Sussman [1] Molecular Tour β-Secretase (β-site amyloid precursor protein cleaving enzyme 1; BACE1) is a transmembrane aspartic protease that cleaves the β-amyloid precursor protein en route to generation of the amyloid β-peptide (Aβ) that is believed to be the principal component of the amyloid plaques seen in the brains of Alzheimer’s disease. It is thus a prime target for the development of inhibitors that may serve as drugs in the treatment and/or prevention of Alzheimer’s disease. The protease domain has a conserved aspartic protease fold, with the substrate-binding cleft located between the N- and C-terminal lobes (colored cyan and yellow, respectively). The conserved catalytic Asp dyad, Asp32 and Asp228, is located at the interface of the two lobes. The hairpin loop in the N-terminal lobe that is known as the ‘flap’ (residues 67-75) partially covers the cleft, and is perpendicular to it, as is in general the case for the aspartic protease family. Determination of the crystal structures of both apo and complexed BACE1, structural analysis of all crystal structures of BACE1 deposited in the PDB, and MD simulations of monomeric and ‘dimeric’ BACE1 were used to study conformational changes in the active-site region of the enzyme (the first monomer in magenta with a red flap, the second one in green with an orange flap). Four new crystal structures were determined: that of a BSIIV/BACE1 complex belonging to space group P6122 (3tpr, BACE1 in magenta and BSIIV in cyan), those of a second BSIIV/BACE1 complex (3tpp, BACE1 in indigo and BSIIV in green) and of an apo structure (3tpj, in orange), both belonging to space group C2221, and that of a second apo structure belonging to space group C2 (3tpl, in blueviolet). The crystals of the BSIIV complex belonging to space group P6122 (3tpr, BSIIV/BACE1) were obtained by soaking the inhibitor BSIIV into a crystal of the apo enzyme belonging to the same space group, while those of the complex belonging to space group C2221 were obtained by cocrystallization of the inhibitor and the enzyme (3tpp, BSIIV/BACE1). The two apo structures belonging to different space groups were obtained by cocrystallization in the presence of another low affinity inhibitor, using two different precipitants; in both cases the inhibitor was not seen in the crystals thus obtained. It was found that the conformations of the enzyme in the two BSIIV/BACE1 crystal structures differ significantly from each other, as do the two apo structures, particularly in the active-site flap and in the 10S and 113S loops. This led to a systematic analysis of all the BACE1 crystal structures deposited in the PDB (both apo structures and complexes), and to perform MD simulations on the apo enzyme, so as to investigate the underlying causes for the various conformations adopted by the flap. In particular, an attempt was made to clarify whether they might be a consequence of crystal packing in different space groups, or might indicate that the active site can assume multiple low-energy conformations. The RMSFs of main-chain atoms, calculated based on all the BACE1 crystal structures deposited in the PDB, show that the flap is the most flexible region of the protein. The minimal distance between the flap residues and the catalytic residue D228, which provides a measure of the extent of opening of the flap, reveals that the flap adopts an open conformation in all the apo structures (e.g. 1w50, colored deepskyblue) with two exceptions; not surprisingly, the situation with respect to the structures of the complexes is more complex. In most of the complexes, direct H-bond interaction between main chain atoms of flap residues and the bound inhibitor lock the flap in the closed conformation (e.g. 3tpp, BSIIV/BACE1), except in a few cases in which the position and orientation of the inhibitors sterically preclude its closing. It is of interest that no direct H-bonds are formed between the inhibitor and the flap in complexes in which the flap is in a more open conformation, as is the case in the crystal structure of the complex obtained by soaking BSIIV into apo crystals of BACE1 (3tpr, BSIIV/BACE1); but a direct H-bond between Q73 and BSIIV locks the flap in a closed conformation in the co-crystallized structure (3tpp, BSIIV/BACE1). It may thus be concluded that formation of a direct H-bond between a main-chain atom of a flap residue and the bound inhibitor is a prerequisite for the flap to assume a closed conformation in a complex. So, there are three possible conformations of flap - locked, open and intermediate. Interestingly, in all the crystal structures of complexes that belong to space group P6122 except one, the flap is in an open conformation. Our soaked crystals of the BSIIV/BACE1 complex also belong to space group P6122. Analysis of the crystal packing pattern reveals that in this space group the flap is at a packing interface. MD simulations were carried out to check if the observed packing would influence the conformation of the flap. In the starting model for MD that includes a symmetry-related copy of BACE1, both the degree of opening and the time that the flap dwells in the open state are much larger than in the trajectories generated for the monomer. Thus, the packing pattern in crystals belonging to space group P6122 hinders the conformational change in the flap, which may be the main reason why in our BSIIV/BACE1 complex (3tpr) belonging to space group P6122 the bound BSIIV does not cause the flap to adopt a closed conformation, whereas it does so in the complex belonging to space group C2221 (3tpp, BSIIV/BACE1). Thus a systematic study of the conformational flexibility of BACE1 may not only contribute to structure-based drug design of BACE1 inhibitors, but also produce a better understanding of the mechanistic events associated with binding of substrates and inhibitors to the enzyme. PDB references: BACE1, apo, 3tpj; 3tpl; BSIIV complex, 3tpp; 3tpr. References
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