Beta-amyloid precursor protein cleavage enzyme 1 (BACE1) and beta-amyloid precursor protein cleavage enzyme 2 (BACE2), members of aspartyl protease family, are close homologues and have high similarity in their protein crystal structures. However, their enzymatic properties differ leading to disparate clinical consequences. In order to identify the residues that are responsible for such differences, we used evolutionary trace (ET) method to compare the amino acid conservation patterns of BACE1 and BACE2 in several mammalian species. We found that, in BACE1 and BACE2 structures, most of the ligand binding sites are conserved which indicate their enzymatic property of aspartyl protease family members. The other conserved residues are more or less randomly localized in other parts of the structures. Four group-specific residues were identified at the ligand binding site of BACE1 and BACE2. We postulated that these residues would be essential for selectivity of BACE1 and BACE2 biological functions and could be sites of interest for the design of selective inhibitors targeting either BACE1 or BACE2.
Members of aspartyl protease family are known to be associated with some pathological states such as breast cancer and pruritic inflammatory skin disease [
BACE1 is a type 1 transmembrane protein which is highly expressed in brain and pancreas, and it can also be found in other organs at much lower levels [
Bioinformatics tools have been widely used for predicting protein functions. Various computational methods for predicting protein structure and functions have been developed. Methods to predict protein functions can be divided into sequence-based method and structure-based method [
Human BACE1 and BACE2 sequences with UniProt accession numbers P56817 and Q9Y5Z0, respectively, were used as query sequences for BLASTP [
Multiple sequence alignment was carried out on the selected sequences using ClustalW [
Evolutionary trace based cladogram of selected BACE1 and BACE2 protein sequences from UniProt database. ET partition cut-off divides the phylogenetic tree into Group 1 (BACE1) and Group 2 (BACE2).
Then, the sequences were separated based on groups. Sequences within each group were separately aligned together and consensus sequences were obtained for a particular group. The consensus sequences were classified as neutral, group-specific, and conserved residues. Conserved residues are amino acid residues that are conserved in the multiple sequence alignment while neutral residues are amino acid residues that are not conserved. Group-specific residues are amino acid residues that are conserved within a particular group but differ from other groups. The consensus sequences were then compared and an ET sequence was derived. ET sequences were compared to the query sequences (P56817, Q9Y5Z0). To identify the location of conserved and group-specific amino acid residues, the ET sequence was aligned with the query sequences (Figure
ET sequence compared to the seed sequences of BACE1 (P56817) and BACE2 (Q9Y5Z0). The domains within BACE1 and BACE2 sequences are marked with the coloured thick lines. Red and blue lines are for the N-terminal and C-terminal domains, respectively, whereas the green lines marked the interdomain regions within the structures. Amino acid residues for the flap region are marked within the box. Conserved residues are shown as their specific amino acid in the ET sequences, while group-specific residues are shown as alphabet “X.” Neutral residues are marked as “-” in the ET sequence. The amino acid numbering of BACE1 and BACE2 sequences is according to their 3D structures obtained from PDB file, 1FKN, and 2EWY, respectively. The starting amino acid residues are highlighted in red. Amino acid residues that are involved in the ligand binding sites of BACE1 and BACE2 are marked with asterisk. Red coloured asterisks are binding sites that are only for BACE1, while blue coloured asterisks are the ligand binding sites that are only for BACE2.
Crystal structures of human BACE1 and BACE2 with the PDB codes 1FKN and 2EWY, respectively, were retrieved from Protein Data Bank (PDB) [
We analyzed BACE1 and BACE2 protein sequences from several mammalian species using ET method to identify the functional sites that lead to different functional properties of BACE1 and BACE2. Developed by Lichtarge et al. in 1996, ET method enables the identification of conservation pattern within homologous proteins by comparing both the amino acid sequence and protein crystal structure information [
Our ET analysis revealed that 189 amino acid residues were conserved which is 37.7% of human BACE1 and 36.4% of human BACE2. Furthermore, 123 group-specific residues were identified, which comprise 24.5% of human BACE1 and 23.7% of human BACE2.
BACE1 and BACE2 are close homologs that not only share sequence similarity, but also have very similar 3D structure. Overall structures of BACE1 and BACE follow the general fold of aspartyl protease family comprising an N-terminal domain, a C-terminal domain, and an interdomain that connects the N-terminal and C-terminal domains (Figure
Chain A of crystal structures of BACE1 (a) and BACE2 (b). In both structures, the N-terminal, inter-, and C-terminal domains are coloured blue, yellow, and green, respectively. The ligands are displayed with “Ball and Stick” and red colour.
Superimposition of the crystal structures of BACE1 (1FKN) and BACE2 (2EWY) gave a root-mean-square deviation (RMSD) of 1.46 over 373 C-alpha atoms indicating that the structures of BACE1 and BACE2 are very similar to each other (Figure
Superimposition of chain A of crystal structures of BACE1 (1FKN) and BACE2 (2EWY), where BACE1 is coloured blue and BACE2 is coloured green.
We defined the ligand binding site as all amino acid residues within 5 Å from the ligand. Twenty-eight of amino acid residues in BACE1 and 24 of amino acid residues in BACE2 were identified as ligand binding site. The ligand binding site residues of BACE1 and BACE2 together with their evolutionary trace status are summarized in Table
Amino acid residues at the ligand binding sites of BACE1 and BACE2. The amino acid residue numbering is according to their respective 3D structure (1FNK and 2EWY).
BACE1 | BACE2 | ||
---|---|---|---|
Amino acid residues | Trace status | Amino acid residues | Trace status |
Ser10 | Conserved | ** | — |
Gly11 | Conserved | ** | — |
Gln12 | Conserved | ** | — |
Gly13 | Conserved | ** | — |
Leu 30 | Conserved | Leu46 | Conserved |
Asp32 | Conserved | Asp48 | Conserved |
GLy34 | Conserved | Gly50 | Conserved |
Ser35 | Conserved | Ser51 | Conserved |
Val69 | Conserved | Val85 | Conserved |
Pro70 | Group-specific | Lys86 | Group-specific |
Tyr71 | Conserved | Tyr87 | Conserved |
Thr72 | Conserved | Thr88 | Conserved |
Gln73 | Conserved | Gln89 | Conserved |
* | — | Gln90 | Conserved |
Phe108 | Conserved | Phe124 | Conserved |
Ile110 | Group-specific | Leu126 | Group-specific |
Trp115 | Conserved | Trp131 | Conserved |
Ile118 | Conserved | Ile134 | Conserved |
Ile126 | Group-specific | Leu142 | Group-specific |
Tyr198 | Conserved | Tyr211 | Conserved |
Lys224 | Conserved | ** | — |
Ile226 | Conserved | Ile239 | Conserved |
ASP228 | Conserved | Asp241 | Conserved |
Gly230 | Conserved | Gly243 | Conserved |
Thr231 | Conserved | Thr244 | Conserved |
Thr232 | Conserved | Thr245 | Conserved |
Asn233 | Group-specific | Leu246 | Group-specific |
Arg235 | Conserved | Arg248 | Conserved |
Thr329 | Conserved | ** | — |
* | — | Ser337 | Conserved |
*Ligand binding site not identified in BACE1.
**Ligand binding site not identified in BACE2.
We mapped the ligand binding sites and their ET status onto the chain A of crystal structures of BACE1 (1FKN) and BACE2 (2EWY) (Figure
The display of ligand, conserved, and group-specific residues within the ligand binding sits of BACE1 (a, b) and BACE2 (c, d). The ligands are coloured red while conserved and group-specific residues are coloured cyan and purple.
Members of aspartyl protease family are known to have a loop structure that covers the active site of enzyme upon binding with the ligand. This structure is known as the flap region, and it is flexible and can adopt different structural conformations upon binding with the substrate. It is assumed that this structure will shield the active site from the solvent [
The last group-specific residue at the ligand binding site is Asn233 of BACE1 substituted with Leu246 of BACE2. The observation indicated that, in BACE2, the binding site at this region is more hydrophobic than the one in BACE1. This difference can be exploited for the design of a selective drug targeting either BACE1 or BACE2.
Designing a drug that could selectively inhibit BACE1 could avoid the unwanted side-effects of also inhibiting other aspartic protease family members including BACE2. BACE1 and BACE2 are close homologs and they are competing against each other for the same substrate (A
ET analysis on amino acid sequences and protein structures of BACE1 and BACE2 from several mammalian species enabled us to identify the distinctive features of BACE1 and BACE2 amino acid sequences. Mapping the ET analysis onto a known 3D structure of BACE1 and BACE2 revealed that their active sites are well conserved. Four group-specific residues were identified in the ligand binding sites of BACE1 and BACE2. The residues are Pro70, Ile110, Ile126, and Asn233 of BACE1 substituting Lys86, Leu126, Leu142, and Leu246 of BACE2, respectively. These group-specific residues would be the reason for cleavage site selectivity in BACE1 and BACE2 biological function and would be the potential residues for the design of selective and specific inhibitors targeting either BACE1 or BACE2.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank Director General of Ministry of Health Malaysia for permission to publish this paper. This research is cosupported by the High Impact Research Grant UM-MOHE UM.C/625/1/HIR/MOHE/SC/30 from the Ministry of Higher Education Malaysia and University of Malaya Research Grant (UMRG), Grant no. RP004C-13AFR.