Beta amyloid Precursor Complexed with Trypsin

Patty Hunt, Lisa Jungbauer, Laura Melohn

One event leading to Alzheimer’s Disease (AD) is the aggregation of beta amyloid protein (BAP) to form neural plaques or lesions, which leads to neural cell death. BAP is the major component of cerebral amyloid deposits in Alzheimer’s disease, and is derived from the cleavage of beta-amyloid precursor protein (BAPP) into a 40-42 amino acid residue in the extracellular domain.¹   BAPP exists in two regions, as a transmembrane protein across the lipid bilayer of neural cells (refer to figure 1)² and in the extracellular fluid of the central nervous system and blood. Three isoforms of BAPP are formed when BAPP mRNA is alternatively spliced resulting in two isoforms containing Kunitz proteinase inhibitor (KPI) domain and one without the domain.³             

Figure 1  BAPP as a Transmembrane Protein

The KPI domain occurs around the region of residues 289-342 of BAPP, ⁴ is comprised of 56 amino acids, and inhibits the activity of the serine proteases (trypsin and chymotrypsin).⁵   Overproduction of the BAPP isoforms containing the KPI domain exists in AD patients and prevents normal degredation of BAPP³ which is a suggested route leading to deposition of beta amyloid protein causing sporadic AD.⁶

The KPI domain of BAPP also acts as an anticoagulant in vitro, inhibiting coagulation factors XIa and IXa.⁴

^{Familiarizing Yourself with the BAPP-Trypsin Complex}

• ^{The rendering of the complex (wireframe view, space filling view, and cartoon view) can be altered by using the command buttons on the right.}  
• ^{The image can be zoomed in or out by holding down the shift key and the left mouse button while dragging the mouse.}
• ^{The backbone of the complex can be viewed by right clicking on the complex image (with MDL written in the corner) above.  Select display and backbone.  The rendering of only the backbone allows for the two molecules to be easily distinguished (BAPP is the smaller elongated molecule and Trypsin is the large globular molecule.}
• ^{The hydrophobic areas of the complex can be viewed by right clicking on the image and selecting select, protein, and  hydrophobic.   To display the change right click on the image, select display and ball and stick.  All ball and stick representations show the hydrophobic portions of the complex.} 
• ^{The charged portions of the complex can be viewed using the same actions as the hydrophobic areas only select charged instead of hydrophobic.} 
• ^{The solvent accessible areas of the complex can be viewed by right clicking on the image, selecting options, dot surface, and Connely/Richards solvent.} 

First, consider the KPI domain of BAPP.

Load Uncomplexed Beta Amyloid Precursor Protein

BAPP free in solution exists as a dimer. BAPP has two binding loops that form the interface with the target protease and there are hydrogen bonds between the backbone atoms of the two proteins.  The ribbon model facilitates viewing of the two binding loops.

Ribbon View

Original Settings

MET 17 (methionine) and PHE 34 (phenylalanine), located at the end of the second protease binding loop, create a large hydrophobic patch at the surface of the interface (see figure 2).

Figure 2: Hydrophobic patch of BAPP

The space filling model is the MET-17 and the amino acid marked CB_2:34 is the PHE-34.

The hydrophobic patch is an optimal site for proteins with surface hydrophobes to interact. Trypsin does not contain hydrophobic residues that would correspond to the hydrophobic patch in BAPP, which lowers its binding affinity, however BAPP does bind trypsin well with K_I = 10^-10 M.

Now consider the BAPP-trypsin complex. The importance of the binding interaction remains somewhat undetermined and controversial but is suspected to be involved in proteolytic events leading to cerebral amyloid deposition.³

Load Complexed Beta Amyloid Precursor Protein

The primary specificity residue of BAPP is an ARG (residue 15) located at the center of the first (of two) protease binding loops.³ ARG is a basic amino acid with a positive charge at physiological pH. ARG forms a salt bridge with ASP 189 of the trypsin, which is negatively charged at physiological pH. Figure 3 illustrates the salt bridge interaction deep within the trypsin active site. 

Figure 3: Salt Bridge Interaction of the Complex

The space filling model in the figure above represents the ASP-189 in the trypsin and the amino acid marked with C2_2:15 is the ARG-15 on the BAPP. 

A Ramachandran plot of the complex (figure 4) was generated to depict the frequency that secondary structure occurs in the protein complex. a -helices fall at –47 and 57 in the plot, and beta -sheets fall at –119 and +113 (parallel) or –139 and +135 (antiparallel). The BAPP-trypsin complex contains primarily beta -sheets, which corresponds to a cartoon representation of the complex (figures 5a and 5b).

Figures 5a and 5b: Secondary and Super-secondary Structure

Trypsin is the molecule on the right and BAPP is on the left.

References

1.  Dr. Henry Jakubowski, College of St. Benedict/St. John's University, Chemistry Department 1999.

2.  Price, D., Sisodia, S., Borchelt, D. Genetic Neurodegenerative Diseases: The Human Illness and Transgenic. Science. 1998 November 6; 282: 1079-1083.

3.  Hynes, T., Et al. X-ray Crystal Structure of the Protease Inhibitor Domain of Alzheimer's Amyloid Beta-Protein Precursor.  Biochemistry.   1990 29 (43):

     10018-10022.

4.  Van Nostrand, W, Et al.  Enhanced Plasmin Inhibition by a Reactive Center Lysine Mutant of the Kunitz-type Protease Inhibitor Domain of the Amyloid

     Beta-Protein Precursor. Journal of Biological Chemistry.  1995 Sep 29;270(39):22827-22830.

5.  Ho, L, Fukuchi, K, Younkin, S.  The Alternatively Spliced Kunitz Protease Inhibitor Domain Alters Amyloid Beta Protein Precursor Processing and Amyloid

     Beta Protein Production in Culture Cells.   Journal of Biological Chemistry.  1996 Nov 29;271(48):30929-30934.

6.  Moir, R, Et al.  Relative Increase in Alzheimer's Disease of Soluble Forms of Cerebral Abeta Amyloid Protein Precursor Containing the Kunitz Protease

     Inhibitory Domain.  Journal of Biological Chemistry.   1998 Feb 27;273(9):5013-5019.