Aims

• To give practice in using molecular visualisation software.
• To show different ways in which serine proteases can be inhibited.
• To introduce the idea of designing drugs that inhibit enzymes.

Objectives

After this practical you will:

• be competent in performing simple interactive tasks with the RasMol molecular visualisation program;
• be aware of the catalytic triad of serine protease enzymes;
• be aware of different ways in which serine proteases can be inhibited.

Exercises

1. Protein Data Bank entry 1TLD is a high resolution structure of bovine beta-trypsin, a serine protease. His57, Asp102 and Ser195 are important active site residues in serine protease enzymes. Locate these three residues and draw a sketch showing the distances between the oxygen and nitrogen atoms in their side chains.
   To see the side chains of these residues more easily, display the whole structure as a wireframe model, then select the three side chains:

         RasMol> select (57, 102, 195) and sidechain
       

   Select "Ball & Stick" from the Display menu to highlight these side chains.

2. The mechanism by which serine proteases cleave peptide bonds is illustrated in this animation.

   Note the Asp residue at position 189. Locate this residue in the binding pocket of structure 1TLD. A positively charged side chain from the substrate (the protein chain to be cut) will bind in this pocket.

3. Protein Data Bank entry 3BTK is a complex between bovine beta-trypsin and bovine pancreatic trypsin inhibitor (BPTI). Which BPTI side chain is closest to Asp189 of bovine beta-trypsin? Is the active site of beta-trypsin accessible to a substrate? Try the following sequence of RasMol commands:

         RasMol> colour chain
         RasMol> select 57e,102e,195e
         RasMol> colour cpk
         RasMol> spacefill
         RasMol> select not hoh
         RasMol> spacefill
       

4. Serpins are a family of serine protease inhibitors whose mechanism is different to that of BPTI. Protein Data Bank entry 1ATU is the active form of human alpha-1-antitrypsin, a serpin. Look at this structure, and locate residues Met358 and Ser359. These residues are in the reactive-centre loop, and the reaction with a serine protease cleaves the loop between these two residues. Describe the structure of the main chain between Lys343 and Met358.

5. Protein Data Bank entry 1EZX is a serpin-protease complex. The molecules present are human alpha-1-antitrypsin (a serine protease inhibitor, or serpin) and bovine trypsin (a serine protease). The peptide bond between Met358 and Ser359 of alpha-1 antitrypsin has been cleaved. Measure the distance between the main chain carbon atom of Met358 and the nitrogen atom of Ser359. Describe the structure of the main chain between Lys343 and Met358. Locate the active site residues of the trypsin molecule.

6. Thrombin is a serine protease. Protein Data Bank entry 1BHX shows thrombin and a designed thrombin inhibitor. Highlight the catalytic triad, the aspartic acid in the binding pocket (the S₁ pocket) and the inhibitor (chain L) as follows:

         RasMol> restrict protein 
         RasMol> colour cyan
         RasMol> spacefill
         RasMol> select (his57, asp102, ser195) and sidechain
         RasMol> colour cpk
         RasMol> select asp189 and sidechain
         RasMol> colour gold
         RasMol> select *L
       

   Choose "Sticks" from the Display menu to see the inhibitor.

   Compare the 3-D structure in RasMol with the schematic illustration in Figure 4 of Wagner et al. (1998).

Supplementary Material

In October 2003 Trypsin was featured as the Protein Data Bank's Molecule of the Month and was the European Bioinformatics Institute's Protein of the Month. In May 2004 Serpins were featured in the Molecule of the Month series.

You can find out more about the serpin-protease complex in the article describing the structure:

• Huntington, J.A., Read, R.J., Carrell, R.W. (2000) Structure of a Serpin-Protease Complex Shows Inhibition by Deformation, Nature, 407, 923-926 (PubMed)

Thrombin has also been featured in the Molecule of the Month series.

The structure of thrombin and a designed thrombin inhibitor (1BHX, Question 6) is described in:

• Wagner, J., Kallen, J., Ehrhardt, C., Evenou, J.P. and Wagner, D. (1998) Rational design, synthesis, and X-ray structure of selective noncovalent thrombin inhibitors, J. Med. Chem., 41, 3664-3674 (PubMed)

Recent research on the design of thrombin inhibitors is reviewed in:

• Schwienhorst, A. (2006) Direct thrombin inhibitors - a survey of recent developments, Cell. Mol. Life Sci. (PubMed)