Analysis of protein structure using Kinemage molecular graphics

Author: Prof. Mariusz Jaskólski

Aims

Purpose of this exercise

In this exercise you will learn how to use a relatively simple but very useful interactive molecular graphics illustration tool called Kinemage (from Kinetic Image) and the associated program called Mage. You will use Kinemage to analyze the conformation of the catalytic center of the enzyme carboxypeptidase A and its motion during substrate binding.

Step 1

Get to knowour molecule

• Using the Internet resources (e.g. Wikipedia, publications in PubMed), assemble the essential information about enzymes with carboxypeptidase activity. In your notepad, describe briefly this activity and write the chemical reaction catalyzed by carboxypeptidase. What is the E.C. number (International Enzyme Classification) of carboxypeptidases?

Step 2

Have a look at carboxypeptidase A structures in the PDB

• Using your experience with the PDB, select the Advanced search option and then Molecule name: carboxypeptidase A. Evaluate this Query. How many hits did you get? Display 100 Results per page.
• In Generate Reports select Structure. Sort the hits according to resolution. (Note that NMR structures, which do not have a defined resolution, are pushed up front. Ignore them, as we are interested in crystallographic structures in this exercise.)
• Select the carboxypeptidase A structure determined at the highest resolution. Note its PDB code.
• Have a look inside this PDB file (Display files icon). Detailed and ordered information about this structure can also be obtained by clicking Methods or Geometry.
• In Geometry, you can also investigate the Ramachandran Plot for this structure.
• Make notes to summarize this part of the exercise:
  • who discovered this structure and when?
  • what is the main literature reference? Note it using the Medline format.
  • what is the resolution of this structure?
  • what are the r.m.s. deviations from ideal bond lengths?
  • what are the R and R-free residuals?
  • what is the space group?
  • what are the unit cell parameters?
  • what is the E.C. number associated with this entry?
• Looking at the statistical parameters above, how would you assess this structure?

Step 3

Have a look at carboxypeptidase A in PyMOL

• Download the selected PDB file with Carboxypeptidase A coordinates into your computer.
• Display the structure using PyMOL.
• Familiarize yourself with the structure. Carboxypeptidase A is a metalloenzyme. Can you identify the metal?
• Investigate the coordination of the metal cation in carboxypeptidase A. You need to display side chains to do that!
• What is the metal coordination? Note the distances and angles of the coordinative bonds.
• What type of coordination would you expect for this metal? Does what we have in carboxypeptidase A make sense?
• How do you think this metal-containing active site is functioning during the hydrolysis reaction? Which ligand of the metal cation is the most important one?
• A nice animation of the catalytic mechanism of carboxypeptidase A can be found at this site http://juang.bst.ntu.edu.tw/BCbasics/Animation1.htm#CPA

Step 4

Advanced use of Kinemage

• Open the Mage program by clicking on its icon on the computer Desktop.
• In the File menu of Mage open the kinemage file kin.kin. It is available on your computer Desktop and can be also downloaded from the following URL (Universal Resource Locator): http://www.man.poznan.pl/CBB/CWICZENIA/kin.kin
• kin.kin contains a complicated tutorial, with several Kinemage scripts combined into one file. When it is loaded, you should see Kinemage #1 highlighted in the small upper-right panel. (There are six separate Kinemages there. Later,you may want to have a look at Kinemage #5 as well.)
• A Kinemage script opens not only a graphics window but also a text window. In this text window, under {KINEMAGE 1}*, you will find detailed instructions for the next step of this exercise.

Step 5

Motion in the active site of Carboxypeptidase A

• This animated Kinemage compares the conformation in the active site of carboxypeptidase A during binding of a substrate, the dipeptide glycyltyrosine (Gly-Tyr)
• What happens with this substrate during the enzymatic reaction? Write the reaction equation. Logically, what is being cleaved off from the "rest" of the substrate?
• The change of the conformation of the active site can be observed because we have the crystal structures of the enzyme in both states: before binding the substrate (PDB code 5CPA) and in complex with the Gly-Tyr substrate (3CPA). Both structures were solved by Lipscomb.
• Find those structures and their characteristics in the list of your "carboxypeptidase A" PDB hits.
• The Animation button shows what is happening during substrate binding:
  • The substrate Gly-Tyr (red) appears in the active site
  • Tyr248 from the enzyme (blue) undergoes a huge conformational change
  • the main chain of the 240-253 loop (white), which carries the Tyr248 residue, also undergoes slight conformational adjustments
• Try to visualize the changes (Animate) using different viewing directions.
• The Display menu also has the Stereo on option. Try it!

Step 6

Geometry measurements

• In the Tools menu there are some useful options, e.g. XYZ point displaying the coordinates of the highlighted atom, or Measures, which we will use now to analyze the geometry of the active site of carboxypeptidase A and its changes on substrate binding.
• Determine the magnitude of the movement of the OH atom of Tyr248 on substrate binding.
• Select for yourself one of the conformations of the Tyr240-Gly253 fragment. Your task will be now to measure its geometrical parameters, in particular the torsion angles of the main chain in order to determine the type of polypeptide chain fold.
• Switch off all the unnecessary options, such as Tyr248, other sc, GlyTyr.
• Before starting the measurements, open the location http://www.man.poznan.pl/CBB/CWICZENIA/CHEM/SERP-3/table.txt to find a useful template for noting your measurements.
• Copy the table.txt template to an editor. Use proportional font, for instance Courier.
• You will be measuring the bond distances, bond angles and torsion angles of the main chain of the Tyr240-Gly253 fragment.
• The best way to do this is to "walk" along the main chain starting from the N atom of Tyr240, and clicking successively the connected atoms of the main chain.
• Activate the Measures option from Tools.
• When you click an atom, its label appears at the bottom of the graphics window. (NOTE: sometimes the carbonyl C atoms are incorrectly labeled as "P".)
• When you click another atom, you will see its distance from the previously clicked atom. A soon as a third atom is clicked, you will also see the angle defined by those last three atoms. Finally, as soon as you have clicked a fourth atom, it will define a torsion angle and the corresponding value will be displayed.
• You should be noting the results of your measurements in table.txt as they appear during your "walk" along the main chain.
• The first numbers have been already inserted into table.txt. Please complete the table for values from Tyr240 to Gly253. You will quickly see the logic of this table: a bond distance is next to the | bond, then a bond angle next to an atom which defines its apex, finally the torsion angle is typed again in the line containing the | bond, on which it is centered.
• Please work very carefully, especially selecting the "next" atom. If you make a mistake, you will have to start from the beginning.

Step 7

Analysis of the results

• You should be able to check (and if necessary - find) any errors or mistakes in the results of your measurements.
• The first check is very simple, a visual inspection. Because of the repetitive nature of the polypeptide chain (...-N-CA-CO-N-CA-CO-...), you should see in the columns of your numbers the same repeating pattern. Look for a pattern of three repeating values in the Bond column.
• An even easier check is possible in the Torsion angle column, because here every third angle is the peptide omega dihedral angle, which should be trans, i.e. close to 180 degrees.
• Now extract from your table the phi/psi pairs. Copy them in separate columns of an Excel spreadsheet (i.e. the phi angles should be put in one column, and the matching psi values in the same row of the next column).
• Use the Plot function of Excel to construct a diagram representing the Ramachandran Plot. Remember that the the phi angle on the horizontal axis spans the interval from -180 to +180 degrees. A similar interval is covered by the psi angle along the vertical axis. It means that the origin of your Ramachandran Plot will be in the center of the square plot area.
• When "your" Ramachandran Plot is ready, try to figure out what was the secondary structure (conformation) of the Tyr240-Gly253 fragment of carboxypeptidase A that you have been analyzing.
• Write your results and observations in your report.

Step 8

Your Report

• Your Report from this practical, as from all other practicals, should be completed and sent to your tutor by email (szymon@amu.edu.pl) before the date of the following practical class.

Some extra fun...

• If you have finished this exercise with Kinemage #1 quickly, you can open Kinemage #5 which illustrates the Leucine Zipper motif of two alpha-helices, winding gently in a left-handed manner around each other in a superhelical, or coiled-coil fashion. Try to appreciate and enjoy this beautiful and highly logical motif!

Homework

Next week, you will be studying the enzyme L-asparaginase. You should familiarize yourself with this subject by reading the following review article:

• Michalska K, Jaskolski M. 2006. Structural aspects of L-asparaginases, their friends and relations. Acta Biochim. Polon. 53: 627-640. (PDF)

In particular, you will be analyzing a very unusual cluster of aspartates and calcium ions that was found inside the crystal structure of the enzyme EcAIII, which is a type-III L-asparaginase from the bacterium Escherichia coli. Your work will included analysis of electron density maps of EcAIII. To be able to understand the next exercise properly, you should read in advance section 3.5 of the following article:

• Michalska K, Borek D, Hernandez-Santoyo A, Jaskolski M. 2008. Crystal packing of plant-type L-asparaginase from Escherichia coli. Acta Cryst. D64: 309-320. (PDF)

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Mariusz Jaskolski, 28.03.2011