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BACE1BACE1

BACE1 (β-site of APP cleaving enzyme) also called β-Secretase and memapsin-2 is a 52 kD class I transmembrane aspartic acid protease that cleaves the Amyloid Precursor Protein (APP) in a rate limiting step that contributes to the accumulation of β-amyloid plaques in Alzheimer’s disease (AD). A subsequent cleavage by γ-secretase generates a 40 or 42 amino acid β-amyloid peptide. These peptides can form Aβ plaques that may have deleterious effects on neuronal function and contribute to pathologies of AD. Under normal conditions, BACE1 activity generates a monomeric and soluble Aβ peptide that may play a physiological role in decreasing excitotoxicity and neurotransmission at glutamatergic synapses. Additionally, α-secretase and γ-secretase cleave APP to generate p3 and the carboxy terminal fragment AICD in a non-amyloidogenic pathway. In AD, amyloidogenic pathways become preferential over non-amyloidogenic and Aβ plaques appear under increased levels of BACE1 catalytic activity.

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The structure of BACE1 catalytic coreThe structure of BACE1 catalytic core

PDB ID 1fkn

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1fkn, resolution 1.90Å ()
Non-Standard Residues:
Activity: Memapsin 2, with EC number 3.4.23.46
Resources: FirstGlance, OCA, PDBsum, RCSB
Coordinates: save as pdb, mmCIF, xml


BACE1 is a bilobal protein that consists of luminal N-lobe and C-lobe anchored to the plasma membrane by a transmembrane domain that exposes a small cytosolic domain. The catalytic core of BACE1, shown here, spans amino acid residues 43-385. BACE1 is a member of the mainly beta class of proteins with a β-barrel architecture. This structure is composed of (shown in green and grey, respectively). Unlike other class I aspartic proteases, insertions and an extension in the C-terminus of BACE1 creates an enlarged C-terminal lobe. are localized on the surface of the C-lobe adjacent from the N-terminus of bound inhibitors. Four acidic amino acid residues on the F insertion loop form a negatively charged area on the surface of the enzyme. are located on the surface of the C-lobe near the C-terminus of bound inhibitors. These BACE1 insertions may form interactions with cell surface components and enlarge the C-terminal surface area to make the active site more accessible to substrates[1]. A 35 residue C-terminal (359-385) forms an ordered β structure spanning residues 369-376 and a helix between residues 378-383. This structure may form a stem with the transmembrane domain. BACE1 contains three structural disulfide bonds. The positioning of these disulfide bonds within BACE1 varies greatly from the location of disulfide bonds in other Class I aspartic proteases[2]. The three structural disulfide bonds maintain the correct fold of BACE1 but are not involved in BACE1 catalysis[3][4].

Structural elements involved in catalysisStructural elements involved in catalysis

PDB ID 1w50

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1w50, resolution 1.75Å ()
Ligands:
Activity: Memapsin 2, with EC number 3.4.23.46
Related: 1fkn, 1m4h, 1py1, 1sgz, 1ujj, 1ujk, 1w51
Resources: FirstGlance, OCA, RCSB, PDBsum
Coordinates: save as pdb, mmCIF, xml


An aspartic acid dyad is located in the active site between the N- and C- terminal lobes. Active site are in a coplanar configuration that allows them to coordinate and activate a water molecule. The activated water performs a nucleophilic attack on the peptide carbonyl group of the substrate. Hydrogen bonds between the carbonyl group of the aspartates and the substrate stabilize a geminal diol intermediate. A proton is then transferred from aspartate to the leaving group resulting in peptide bond breakage between Met671 and Asp672 of APP. The products of the hydrolysis reaction leave the active site[5]. The active site of BACE1 is covered by a 10 amino acid flexible antiparallel β-hairpin called the . This flap controls the entrance of substrates into the active site and is in closed conformation in the presence of bound inhibitors and open conformation in the inactive or apo structures. Tyrosine 71 is a conserved residue located at the tip of the flap that undergoes large conformational changes. In apo BACE1 structures, Tyr71 forms intra-flap hydrogen bonds allowing the flap to adopt a more open conformation. In the presence of inhibitor, the phenolic ring of Tyr71 forms weak hydrogen bonds with the substrate causing the flap to adopt a closed conformation. A Cα of 7Å is visible in the inhibitor bound BACE1 complex in comparison to the apo structure[6]. Additionally, the 10s loop undergoes conformational change in the presence of an inhibitor. This loop may confer specificity for the correct substrate.

BACE1 as a therapeutic target for ADBACE1 as a therapeutic target for AD

PDB ID 2zhr

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2zhr, resolution 2.50Å ()
Non-Standard Residues:
Gene: BACE1 (Homo sapiens)
Activity: Memapsin 2, with EC number 3.4.23.46
Related: 2zhs, 2zht, 2zhu, 2zhv
Resources: FirstGlance, OCA, PDBsum, RCSB
Coordinates: save as pdb, mmCIF, xml


BACE1 is a major therapeutic target due it's role in generating Aβ plaques that contribute to AD pathogenesis. There are currently over 70 structures of BACE1 complexed with inhibitors in the protein data bank http://www.rcsb.org/. These studies have not only developed inhibitors that efficiently block BACE1 activity, but also provide a greater understanding of the residues and regions that interact with the substrate in the active site of BACE1. The first inhibitor crystallized in complex with BACE1 was OM99-2. is a transition state analogue inhibitor that is eight residues in length (Glu-Val-Asn-Leu*-Ala-Ala-Glu-Phe) designated P4-P4’ (P4-P3-P2-P1*-P1’-P2’-P3’-P4’). OM99-2 inhibits BACE1 cleavage between the P1*-P1’ bond because of the presence of a hydroxyethylene isotere (*) that cannot be cleaved. The S1 and S3 sites that bind P1 and P3 are hydrophobic pockets[7] wherease sites S2 and S4 contain Arg residues that accommodate residues P2 and P4 of the substrate [8][9]. Additional sites S5-S7 (not shown in this structure) are in the proximity of the insertion helix and may be important for recognition of the substrate [10]. The maintains a closed conformation over OM99-2 through hydrogen bonding between the conserved Tyr71 residue and the substrate at positions P1 and P2' [11][12]. Additionally, a conformational displacement is observed for the in the hydrophobic S3 site of the BACE1 active site upon binding of an inhibitor.

pH dependence of BACE1 activitypH dependence of BACE1 activity

Electrostatic maps of BACE1 structures ranging from pH 4.0-7.0 Structures obtained from Shimizu et al. (2008) Mol. Cell Biol. 28 (11): 3663–3671.

BACE1 is a pH dependent enzyme with optimal enzyme activity at pH 4.5. At this pH, conformational switching of the active site cleft will accommodate the substrate and electron density of catalytic Wat1 is observed in the crystal structure. BACE1 is active at acidic pHs ranging from pH 5.0 to 4.5. At pHs greater than 5, BACE1 structures are in a closed conformation and cannot undergo a conformational transition to bind the substrate[13]. Tyr71 is in a self-inhibiting conformation partially occupying the S1 site in the active site [14]. Below pH 4.5 crystal structures of BACE1 reveal a disordered Wat1 suggesting that this catalytic water is not consistently located in the active site. It is likely that BACE1 employs pH dependence as a dual regulatory mechanism.

Electrostatic maps of crystal structures obtained by Shimizu et al. show that there is a slight increase in positive charge around the flap (blue). Isolated areas around the active site pocket also increase in positive charge. Although there is no crystal structure of APP bound in the BACE1 active site, these areas are near the substrate pockets that are described to bind negatively charged residues of the OM99 BACE1 inhibitor. Therefore, electrostatic charge probably only plays a small and localized role in the BACE1 catalytic mechanism.















Catalytic mechanism of BACE1Catalytic mechanism of BACE1

Catalytic mechanism of BACE1 Modified from Toulokhonova et al. (2003) J. Biol. Chem. 278 (7): 4582–4589.

PDB ID 2zht

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2zht, resolution 2.35Å ()
Gene: BACE1 (Homo sapiens)
Activity: Memapsin 2, with EC number 3.4.23.46
Related: 2zhr, 2zhs, 2zhu, 2zhv
Resources: FirstGlance, OCA, PDBsum, RCSB
Coordinates: save as pdb, mmCIF, xml


BACE1 catalyzes the hydrolysis of the APP peptide bond between Met671 and Asp672 through a general acid-base mechanism carried out by two highly-conserved aspartic acid residues and a that stabilizes a geminol-diol intermediate. BACE1 employs a bi-iso-uni catalytic mechanism predicted to yield two products from one substrate. The enzyme active site contains two catalytic residues, Asp32 and Asp228. Asp32 acts as a general acid while Asp228 acts as a general base with pKas of 3.81 and 9.48, respectively. The free enzyme (E) is monoprotonated. Upon substrate binding (step 1), the loop closes around the substrate forming a tight FHS complex (steps 2). During the third step, the active site water is activated by the basic Asp228. This nucleophilic water is thought to attack the carbonyl carbon of the peptide bond while the acidic Asp32 residue protonates the carbonyl on the substrate resulting in the formation of a tetrahedral intermediate FHA (step 3). Peptide bond breakage and protonation of the leaving amine releases the products (steps 4-5, GHPQ and GHQ) and free enzyme without the active site water (GH) (step 6). Finally, the free enzyme incorporates an active site water restoring catalytic function (step 7)[15].

Unlike other aspartyl proteases, the solvent kinetic isotope effect (SKIE) on catalytic efficiency was small (Kcat/Km) suggesting that proton transfer steps up to the irreversible step (step 4) are not rate limiting. It is important to note that low catalytic efficiency could also be due to multiple proton transfers offsetting one another, loop closure causing water displacement, or formation of an amide hydrate intermediate. Turnover number (kcat), on the other hand, is greatly effected by solvent showing an inverse SKIE. Formation of the tetrahedral intermediate is not rate limited as indicated by no SKIE on catalytic efficiency. The final step is predicted to be the rate limiting step a recharging step where the enzyme is restored to its initial state by regaining a catalytic water molecule. This is supported by the inverse SKIE on turnover of BACE1[16].

Low Barrier Hydrogen BondsLow Barrier Hydrogen Bonds

Low Barrier Hydrogen Bond in the tetrahedral intermediate of BACE1 Modified from Toulokhonova et al. (2003) J. Biol. Chem. 278 (7): 4582–4589.

Previous studies suggest that aspartic acid proteases may contain low barrier hydrogen bonds[17]. Proton NMR studies of free BACE1 protein shows a downfield signal resonating at δ=11.8 ppm. In comparison, the enzyme complexed to the OM99-2 inhibitor reveals another resonance at δ=13.0 ppm. The D/H fractionation factor for this downfield proton chemical resonance was lower (∅=0.6) than that of free BACE1 (∅=2.2). Proton NMR and SKIE data suggests that formation of a short, strong hydrogen bond occurs in tetrahedral intermediate (FHA)[18].







Evolutionary conservation in the asparic protease familyEvolutionary conservation in the asparic protease family

Members of the aspartic protease family have structural features that are evolutionarily conserved and appear in most aspartic proteases. Pepsin, Cathepsin D, Chymosin, and renin are members of the aspartic protease family and are evolutionarily related to BACE1. Like BACE1, these proteins have a bilobal structure, a catalytic dyad composed of two Asp, and have a β-barrel architecture. On the other hand, BACE1 has several unique features that separate it from other aspartic proteases. Currently, BACE1 is the only member of this family that is a transmembrane protein. The addition of extension loops and helices enlarge the BACE1 C-lobe and the location of disulfide bonds varies in comparison to other aspartic proteases.

LinksLinks

  • 1w50 (Apo structure of bace (beta secretase))
  • 1w51 (Bace (beta secretase) in complex with a nanomolar non-peptidic inhibitor)
  • 2zhs, 2zht, 2zhu, 2zhv (Crystal structure of BACE1 at varying pH)
  • 2zhr (Crystal structure of BACE1 in complex with OM99-2 at pH 5.0)
  • 1fkn (Structure of Beta-Secretase Complexed with Inhibitor)

ReferencesReferences

  1. ↑ Hong L, Koelsch G, Lin X, Wu S, Terzyan S, Ghosh AK, Zhang XC, Tang J. Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor. Science. 2000 Oct 6;290(5489):150-3. PMID:11021803
  2. ↑ Bennett BD, Babu-Khan S, Loeloff R, Louis JC, Curran E, Citron M, Vassar R. Expression analysis of BACE2 in brain and peripheral tissues. J Biol Chem. 2000 Jul 7;275(27):20647-51. PMID:10749877 doi:10.1074/jbc.M002688200
  3. ↑ Haniu M, Denis P, Young Y, Mendiaz EA, Fuller J, Hui JO, Bennett BD, Kahn S, Ross S, Burgess T, Katta V, Rogers G, Vassar R, Citron M. Characterization of Alzheimer's beta -secretase protein BACE. A pepsin family member with unusual properties. J Biol Chem. 2000 Jul 14;275(28):21099-106. PMID:10887202 doi:10.1074/jbc.M002095200
  4. ↑ Fischer F, Molinari M, Bodendorf U, Paganetti P. The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity. J Neurochem. 2002 Mar;80(6):1079-88. PMID:11953458
  5. ↑ Shimizu H, Tosaki A, Kaneko K, Hisano T, Sakurai T, Nukina N. Crystal structure of an active form of BACE1, an enzyme responsible for amyloid beta protein production. Mol Cell Biol. 2008 Jun;28(11):3663-71. Epub 2008 Mar 31. PMID:18378702 doi:10.1128/MCB.02185-07
  6. ↑ Patel S, Vuillard L, Cleasby A, Murray CW, Yon J. Apo and inhibitor complex structures of BACE (beta-secretase). J Mol Biol. 2004 Oct 15;343(2):407-16. PMID:15451669 doi:10.1016/j.jmb.2004.08.018
  7. ↑ Stockley JH, O'Neill C. Understanding BACE1: essential protease for amyloid-beta production in Alzheimer's disease. Cell Mol Life Sci. 2008 Oct;65(20):3265-89. PMID:18695942 doi:10.1007/s00018-008-8271-3
  8. ↑ Ostermann N, Eder J, Eidhoff U, Zink F, Hassiepen U, Worpenberg S, Maibaum J, Simic O, Hommel U, Gerhartz B. Crystal structure of human BACE2 in complex with a hydroxyethylamine transition-state inhibitor. J Mol Biol. 2006 Jan 13;355(2):249-61. Epub 2005 Nov 8. PMID:16305800 doi:10.1016/j.jmb.2005.10.027
  9. ↑ Hong L, Koelsch G, Lin X, Wu S, Terzyan S, Ghosh AK, Zhang XC, Tang J. Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor. Science. 2000 Oct 6;290(5489):150-3. PMID:11021803
  10. ↑ Turner RT 3rd, Hong L, Koelsch G, Ghosh AK, Tang J. Structural locations and functional roles of new subsites S5, S6, and S7 in memapsin 2 (beta-secretase). Biochemistry. 2005 Jan 11;44(1):105-12. PMID:15628850 doi:10.1021/bi048106k
  11. ↑ Hong L, Tang J. Flap position of free memapsin 2 (beta-secretase), a model for flap opening in aspartic protease catalysis. Biochemistry. 2004 Apr 27;43(16):4689-95. PMID:15096037 doi:10.1021/bi0498252
  12. ↑ Shimizu H, Tosaki A, Kaneko K, Hisano T, Sakurai T, Nukina N. Crystal structure of an active form of BACE1, an enzyme responsible for amyloid beta protein production. Mol Cell Biol. 2008 Jun;28(11):3663-71. Epub 2008 Mar 31. PMID:18378702 doi:10.1128/MCB.02185-07
  13. ↑ Shimizu H, Tosaki A, Kaneko K, Hisano T, Sakurai T, Nukina N. Crystal structure of an active form of BACE1, an enzyme responsible for amyloid beta protein production. Mol Cell Biol. 2008 Jun;28(11):3663-71. Epub 2008 Mar 31. PMID:18378702 doi:10.1128/MCB.02185-07
  14. ↑ Gorfe AA, Caflisch A. Functional plasticity in the substrate binding site of beta-secretase. Structure. 2005 Oct;13(10):1487-98. PMID:16216580 doi:10.1016/j.str.2005.06.015
  15. ↑ Toulokhonova L, Metzler WJ, Witmer MR, Copeland RA, Marcinkeviciene J. Kinetic studies on beta-site amyloid precursor protein-cleaving enzyme (BACE). Confirmation of an iso mechanism. J Biol Chem. 2003 Feb 14;278(7):4582-9. Epub 2002 Nov 27. PMID:12458195 doi:10.1074/jbc.M210471200
  16. ↑ Toulokhonova L, Metzler WJ, Witmer MR, Copeland RA, Marcinkeviciene J. Kinetic studies on beta-site amyloid precursor protein-cleaving enzyme (BACE). Confirmation of an iso mechanism. J Biol Chem. 2003 Feb 14;278(7):4582-9. Epub 2002 Nov 27. PMID:12458195 doi:10.1074/jbc.M210471200
  17. ↑ Northrop DB. Follow the protons: a low-barrier hydrogen bond unifies the mechanisms of the aspartic proteases. Acc Chem Res. 2001 Oct;34(10):790-7. PMID:11601963
  18. ↑ Toulokhonova L, Metzler WJ, Witmer MR, Copeland RA, Marcinkeviciene J. Kinetic studies on beta-site amyloid precursor protein-cleaving enzyme (BACE). Confirmation of an iso mechanism. J Biol Chem. 2003 Feb 14;278(7):4582-9. Epub 2002 Nov 27. PMID:12458195 doi:10.1074/jbc.M210471200
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