Analysis of the active site of nucleotidyl
transferases using PyMol
Purpose of this exercise. In this exercise you
will learn how to use a highly professional molecular graphics program called PyMol. You will apply PyMol to analyze the
catalytic sites with divalent metal cations of nucleic-acid-processing enzymes
called nucleotidyl transferases.
Step 0
Reading
required in preparation for this practical:
·
W. Yang, J.Y. Lee, M. Nowotny (2006). Making and
breaking nucleic acids: two-Mg2+-ion catalysis and substrate
specificity. Mol. Cell 25, 5-13.
·
M. Jaskolski, J. Alexandratos, G. Bujacz, A. Wlodawer
(2009). Piecing together the structure of retroviral integrase, an important target in AIDS therapy. FEBS J. 276, 2926-2946; sections on "Functional properties of retroviral INs", "The
catalytic domain of IN", "Structural basis of the enzymatic activity of IN" (available here)
Recommended reading:
·
M. Nowotny, S.A. Gaidamkov, R.J. Crouch, W. Yang
(2005). Crystal structures of RNase H bound to an RNA/DNA Hybrid: substrate
specificity and metal-dependent catalysis. Cell
121, 1005-1016.
Step 1
Get to know
PyMol
·
PyMol is a public domain, highly advanced molecular
graphics program developed by DeLano Scientific. PyMol has its own Wiki
resource, and you should first visit this site at www.pymolwiki.org/
·
PyMol manuals are available on-line at http://pymol.sourceforge.net/
·
An excellent site showing advanced uses of PyMol is
maintained by Daan Van Aalten at http://davapc1.bioch.dundee.ac.uk/teach/pymol/
·
When you launch PyMol, you get an External GUI
(Graphical User Interface) (gray window) and a graphics viewer with an Internal
GUI on the right. You can control PyMol by selecting commands from the External
GUI Menu Bar, by typing commands in the command line, or by executing (Run) a script with PyMol commands (for
an example click here). The latter mode is very
useful; it allows you to know exactly what has been done to get a given effect.
Alternatively, a lot of options are available via the Internal GUI, which can
be used to get your picture quickly. In the upper part of the Internal GUI, you
always see different "selections" defined during your session. On
those selections, you can perform Actions (A), Show (S) them in different
style, Hide (H) those displayed styles, work with Labels (L), or with Colors
(C).To work with a given structure, you must always Open it first, e.g. via the File menu.
Step 2
Get to know
our molecule
·
We will look at the active site of an enzyme called RNase H. It is a hydrolase that cuts
RNA chains. RNase H is found in different organisms. It is also a domain of the
reverse transcriptase (RT) of HIV and other retroviruses. When the RT
reverse-transcribes the viral RNA into viral DNA, RNase H is then used to
degrade the now-useless RNA template, so that the complementary DNA strand can
be synthesized. In the second half of the exercise, you will learn about integrase (IN), which is another retroviral enzyme.
Step 3
Have a look
at the PDB
·
In a browser window, open the PDB site.
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Search for HIV RNase H.
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Note the size of the protein, who discovered the
structure, and when.
·
You can already use PyMol to have a look at HIV RNase
H, by a right-button click on the icon near the PDB code, which will allow you
to select the (PyMol) program for opening this PDB entry.
Step 4
RNase H in complex with RNA/DNA and Mg2+
·
Ultimately, we want to analyze the structure of RNase
H from Bacillus halodurans. It has been determined by Nowotny et al. (Cell 121, 2005:1005-1016) in complex with an RNA/DNA hybrid substrate
and magnesium cations (Mg2+), which are necessary for catalysis.
·
Find this structure in the PDB (1ZBL) and display it
using PyMol. Before you proceed any further with this exercise, you should
familiarize yourself with a review article describing the mechanism of
nucleotidyl transferases by Yang et al. (Mol.
Cell 25, 2006:5-13).
Step 5
Get an overview of the 1ZBL structure in PyMol
·
In particular, use the preset simple Action to get a line drawing of the molecule. You
will note that the structure is dimeric, i.e. consists of two copies of the
RNase H subunit. Play with the excellent select/display options of PyMol. For
instance, using the right mouse button, you can select a chain to be shown in a preset stick representation, by just one click!
·
For further work, you might want to simplify the scene
by, for instance, selecting only the protein chain A and hiding the protein
chain B.
·
Find the magnesium ions in the active site. (If you
cannot see them, use this trick: type "select MG, element mg" – a new
selection, MG, pops up; Show it as spheres, you won't be able to miss the Mg2+
cations now!)
·
Select only those residues and those water molecules
which are in the coordination spheres of the Mg2+ cations. Hide
everything else.
·
Find a nice view. You can write out the orientation
matrix by the following commands:
log_open logfile.log opens a logfile
get_view writes out the orientation matrix
log_close closes the logfile
·
Later, you can use this orientation matrix to get
exactly the same view using an analogous set_view command (usually issued from
within a script, as it contains a rather big 3x6 matrix).
·
You can give the selected residues a nice
ball-and-stick representation, for instance, using the Appearance Wizard, which will turn atoms into spheres by simple
clicks. You can control the spheres (like anything else in PyMol) by the
Setting menu Edit All... option.
Editing a given parameter, e.g. sphere_scale or sphere_transparency, will take
immediate effect. Try it!
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In the Mouse menu pick Selection mode as Atoms;
this way you will be selecting only those atoms, on which you click.
·
Click on the two Mg atoms (green crosses) in
succession. A new Selection "(sele)" appears. Change its name under A
to Metal. Working in the same way, select successively all residues (only
side-chain atoms) and water molecules forming the coordination spheres of the
two magnesium cations. For instance, you
might click on the P atom from the RNA backbone which is within the
coordination area, and on its four O atoms, and rename this selection as PO4.
·
You can render each such selection as ball-and-stick
by using again A with preset ball and stick.
·
When our object of interest (the coordination complex)
has been nicely selected and rendered, you might want to hide everything else,
by clicking everything under H
(hide) at 1ZBL.
·
To draw the coordination bonds you will use the Measurement option of the Wizard menu.
·
Now simply click on each pair of atoms that should be
connected by a coordinative bond. (Of course, one point of each such bond will
be one of the Mg atoms!)
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Each selected bond will be displayed as a dashed line
annotated with its length in A.
·
Analyze the coordination spheres of the two Mg2+
cations. What are the coordination numbers? What are the coordination polyhedra?
Which Mg2+ cation has a regular, and which a distorted coordination
sphere?
·
The bond distances are very important, but for a nice
drawing, you may want to delete the annotations. Simply, for each measurement
click on H and hide the label. The coordinative bonds will look
nicer as solid lines. Go again to the Settings window, find "dash"
and edit the following parameters: dash_gap 0, dash_length 1, dash_radius 0.1.
·
To get a beautiful appearance of the drawing, run the
ray tracing procedure by clicking Ray in the External GUI. (Ray tracing is a
special artistic procedure, which handles a three-dimensional object in the
computer by shining light on it and tracking, or tracing, the reflections of
all possible rays from all possible points of the object; in this way one gets
shiny spots, as well as shadows, much as with natural illumination.)
·
If you want to print your figure (Yyyyyyyeeees!!!), it
will look better on white background, which you can select from the Display
menu.
·
The figure can also be printed in stereo. For this
purpose, you have to prepare two drawings (*.png files), one for the left eye
(-left) and one for the right eye (-right). (This is for "wall" or
parallel viewing; if you prefer to cross your eyes, as I do, simply swap the
left/right drawings.)
ray
angle=+3 renders the scene from the left eye's point of view
png left.png writes a file (left.png) with the current scene
ray
angle=-3 renders the scene from the right eye's point of view
png right.png writes a file (right.png) with the current scene
·
You can then paste the left.png and right.png files
into your word or powerpoint processor, or preferably into a paint shop program
such as corel, for printing. For proper viewing (from a distance of 30-40 cm
with normal pupil separation), the components of the stereo pair should be 60
mm apart (distance between equivalent points).
Step 6
Now comes the real thing!
·
Load the PDB file with the atomic coordinates of a
cadmium (Cd2+) complex of the catalytic domain of retroviral
integrase (1VSJ).
·
Intgrase, is the third
(in addition to reverse transcriptase and protease) enzyme encoded by the (RNA)
genome of retroviruses. Retroviral integrase consists of three rather loosely
connected domains: N-terminal domain with a zinc-finger motif, central (core)
catalytic domain, and DNA-binding C-terminal domain. The structure of the
complete integrase is unknown, but we know very well the structure of the
catalytic core domain from very precise crystallographic studies of the protein
form the HIV and Rous sarcoma, or more correctly – Avian Sarcoma, Virus (ASV).
The (catalytic domain of) integrase performs a sequence of important functions.
It binds the cytoplasm-synthesized (by RT) viral DNA and transports it into the
nucleus of the infected cell. In the nucleus, it catalyzes two highly
orchestrated reactions, both of which require DNA cleavage. The first reaction,
called processing, removes two nucleotides from each 3' end of the viral DNA, exposing 3'-OH groups at a
characteristic viral DNA sequence. In the second reaction, called joining, the host DNA is cut on both strands (but at
a characteristic 5-6 bp distance, called stagger), and the exposed 3' ends of
the viral DNA are ligated to the host genomic DNA. The integration is completed
by cellular DNA repair enzymes, which add the 5-6 nucleotides missing at each
integration site as a result of the staggered cut and seal the remaining gaps
(at the free 3' ends of the cut host DNA). Note that through the action of the
integrase, retroviral infection becomes permanent
in the affected cell, as the viral DNA, now called "a provirus", is
coded within the cellular genome! To make the situation even more devastating,
(i) integration can occur at numerous places within one nucleus, (ii) will
additionally lead to damage of the genetic information at the site of
integration, and (iii) can take place at apparently random, non-specific sites
of the host genome. Because the integrase breaks and forms phosphodiester bonds
of the DNA backbone, and in particular because in the joining reaction a DNA
strand transfer takes place, it can be classified as nucleotidyl transferase.
·
Therefore, in the 1VSJ structure we have another
example of nucleotidyl transferase. For its catalytic activity, integrase has
an obligatory requirement for divalent metal cations, manganese (Mn2+)
or magnesium (Mg2+), the latter one being the metal used in vivo. In
experimental conditions, the catalytic domain of retroviral integrase can also
bind other divalent metal cations, such as cadmium, which are useful for
studying the structural aspects of the metal-assisted catalysis of this enzyme.
Step 7
Superposing the two active sites
·
Your task will be now to superpose the active site of
integrase on that of RNase H. It will not be very trivial, but possible. First,
we note some obvious chemical similarities between the two active sites. (i)
Both are arranged around two divalent metal cations. (ii) The separation of the
cations is the same (about 4 A). (iii) The active site is formed by a
constellation of acidic residues; in the retroviral integrase they form the
so-called D,D(35)E sequence signature, with two aspartic acids (D64 and D121 in
ASV integrase) and a glutamate (E157 in ASV), separated from the second
aspartate by exactly 35 residues. (iv) The coordinating ligands are only O
atoms; (v) In both cases, the two metal centers are "bridged" by two
common ligands, by an aspartate (D64 in ASV integrase) and by another O atom:
from a water molecule in the 1VSJ structure (integrase) or from a phosphate
group contributed by the RNA substrate in the 1ZBL structure (RNase H).
·
There is a simple tool in PyMol for superposing two
structures according to best fit of the coordinates of selected atom pairs
(pairs coming from the two structures being aligned, that is). To accomplish
this, select Pair Fitting in the
Wizard menu. A submenu appears in the lower part of the Internal GUI and a
prompt is displayed in the upper left corner of the graphics viewer. Let's say
that we want to superpose the integrase active site (from 1VSJ) onto the active
site of RNase H (from 1ZBL). At each prompt for "mobile atom" select
an atom from the 1VSJ structure and then click on the corresponding atom in the
1ZBL structure ("target atom"). For instance, we can "pair"
each of the Cd2+ ions with their Mg2+ counterparts, and
similarly the O atoms in the bridging carboxylate groups. Each selected pair of
atoms gets connected by a yellow line. Before the fit is calculated the lines
will be pretty long. When you have selected the four atom pairs as described,
you can click on "Fit 4 Pairs" in the Internal GUI submenu. The
superposition matrix will be calculated and the "mobile" structure
will be superposed on the "target" structure. (If during the Pair
Fitting you make a mistake, you can Delete Last Pair or Clear the counter and
start again.)
Step 8
Concluding
·
Have a good look at the results of your work. Do you
think you have chosen the correct Cd-Mg pairs for best correspondence? Or
perhaps the other alternative would be better? Do you think the match of the
active sites is close enough? Is it possible that the catalytic mechanism of
integrase is based on two divalent metal cations in a way similar to that
described for other nucleotidyl transferases? If yes, what would be the roles
of the two metal ions in the active site of integrase?
·
Write down your observations and conclusions.
·
Print a figure with the illustration of your work.
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As a take-home-lesson, remember about the amazing
amount and level of detail of the information
that can be gleaned from three-dimensional structure of macromolecules.
Reflect about the methods by which these structures are determined
experimentally. Reflect about the chemical principles underlying the
functioning of macromolecules, for example coordination of metal ions by
enzymes that break and form P-O bonds in nucleic acids.
Mariusz Jaskolski, 06.12.2009