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ABSTRACT:
Cellular and molecular biology are increasingly concerned with analyzing the three-dimensional
structure of macromolecules and the conformational changes that accompany their function.
Understanding and effectively teaching this material, especially its dynamic features, require
equally dynamic tools. Fortunately, three-dimensional modeling and kinematic visualization are
becoming commonplace features of personal and commercial computing, as evidenced by
graphically complex computer games and numerous movies with dazzling special effects. We are
producing visual aids using current concepts and data (e.g., PDB), various software tools such as
LightWave 3D and Protein Explorer and personal computers with high-performance processors.
Specifically, we have modeled the mechanism of the Na+/K+- pump, relying on the highly
conserved nature of the nucleotide binding domains and phosphorylation sites of P-style pumps
and inferring the structure of its catalytic center from crystallographic data for the SR Ca2+-pump.
Our model simulates the basic features of pump cycling between its E1 and E2 states coupled
with ATP hydrolysis and ion binding and transport. Ouabain inhibition is shown stabilizing the E2
form, while the pump's ability to engage in Na+/Na+ or K+/K+ exchange or to "run backwards" and
synthesize ATP is also simulated. Our models accurately reflect information currently available for
specific transients or the enzymatic cycle; many of the dynamic details, however, are (still)
fanciful. With appropriate rigor concerning folding constraints, however, similar tools could be
used to examine the relationships of primary, secondary and tertiary structures and the possible
dynamics of their transient states. Then, as more data become available, competing hypotheses
could be tested in a dynamic fashion reminiscent of the static manner in which macromolecular
structure were examined with stick models a half-century ago.
Methods and tools:
The most indispensable set of tools we used were the ones that serve to facilitate the use of the
Protein Data Bank. These include Protein Explorer, developed by Eric Martz at the University of
Massachusetts, VMD, developed by W. Humphrey et al. at the University of Illinois and various
PDB search tools, among which PDB Lite, by Eric Martz and Jaime Prilusky (from the Weismann
Institute of Science, Israel), was used the most. Our choice of 3D modeling software was
LightWave 3D by NewTek, but any other package of comparable functionality (for example, 3D
Studio Max by Discreet) would be equally appropriate. We also used AccuTrans 3D by
MicroMouse Productions, Canada for intermediate 3D data translation. These tools, and in
particular the high-end modeling software, require a certain minimum of computing resources.
The Library and Information Services department at Middlebury College provided equipment that
comfortably exceeded this minimum. The setup used for modeling and rendering included an Intel
Xeon processor (2GHz), 2 GB of primary memory and 150 GB of secondary storage space.
Several alternative architectures can be used instead and the performance need not be so high.
The process of generation of the models included three main stages. At first, a manipulatable 3D
structure was generated from the available PDB entries, using spline patching, bone structures
and other 3D modeling techniques. Then, the models were assembled together with auxilliary
elements, such as ATP, the plasma membrane and ions, to form a 3D world of manageable
complexity. Finally, motion was added to this set of models to illustrate the salient scientific theory
about the process modeled. After numerous revisions and a long rendering process the final
product is presented in the form of a digital movie (in our case QuickTime by Apple Computer).
The generation of three-dimensional protein models was the most time-consuming stage of this
project and it involved a lot of extrapolation. Due to the lack of direct information about the spatial
structure of the Na/K pump (best resolution reached is 11Å) we combined two different techniques
for the creation of the models. We built the transmembrane portion of the enzyme in a purely
abstract way, keeping the information conveyed to a minimum and being careful not to include
misleading details. Another consideration was to facilitate the future animation process we had n
mind by creating a suitable model. The beta subunit was modeled in a similar simple and abstract
fashion. The cytoplasmic domains of the Na+/K+ pump, on the other hand, were generated with
the help of three-dimensional information available for the analogous domains of the SR Ca2+
pump (see sequence comparison figures). To further clarify the method, we will follow through the
generation of the shape used to represent the nucleotide-binding domain of the ion pump.
The starting point for the procedure was the PDB file, examined with Protein Explorer. With its
help the sequence of the Ca2+ nucleotide-binding domain was isolated and the binding sequence
visualized. Subsequently, VMD was used to generate a VRML (Virtual Reality Markup Language)
file that reflected the secondary and tertiary protein structure of the isolated domain. This VRML
file was imported into LightWave with the help of AccuTrans 3D, to produce a model ready for
manipulation (see figure 1 - process1Cartoon.gif). In order to simplify this model and make it more
visually pleasing we generated a three-dimensional “skin” for the secondary structure, as
illustrated by the sequence of images (figures 2,3,4 process2CartoonCurves.gif,
process3CartoonWrap.gif, process4FinalSmooth.gif). The process used is called “SplinePatching” and it involves the manual creation of curves (splines) from the existing geometry (fig 2)
and the subsequent generation of a wrapping surface, based on the three-dimensional shape of
the curves (fig 3). After that, a spacefilling model of ATP was used to “carve out” a symbolic
“pocket” where the nucleotide binding sequence was purported to be. Finally, we used
SubPatches, a functionality of LightWave (typical for all 3D modeling software) that allowed for the
generation of a smooth 3D object from the skin generated by the previous steps (fig 4).
In the next part of the process, we used the generated pump models and some other models,
some originating from PDBs (e.g. the plasma membrane, ADP) to populate a three-dimensional
layout where they could all exist and be animated together. After a long period of clarifying details
and consulting various literature, a balance was found between detail and truthfulness of the
models and their movement. As they were intended more as classroom materials rather than
scientific work, an accent was put on keeping them clear and illustrative of the main concepts
involved. Finally, the conceptual layout was solidified into actual animations, presented in the form
of QuickTime movies.
Work in Progress (Topoisomerase)
Another project that we have recently started involves the modeling of the mechanism of action of
E. coli Topoisomerase I. The manipulation of DNA coiling is a process where the threedimensional concept is of great importance and we plan to offer a greater amount of interactivity
in the final presentation. A great help for the development of this model has been the existence of
high-resolution three-dimensional data about the enzyme, thanks to the work of many
researchers, including the teams of H. Feinberg () and L. Yu ().
The first part of the project, generating the topoisomerase model and the DNA model (courtesy of
Gabriel Schine), has been completed. What remains to be done is to conceptualize and assemble
the two components and animate the resultant complex according to the leading theory about the
mechanism of action of the enzyme (illustrated below). Finally, the model will be presented as a
digital movie and as a web-based interactive tutorial.
CAPTIONS:
(from left to right, top to bottom)
To the primary sequence of Na/K pump and the Ca2+ pump domain subdivision
Left top: The domain breakdown of the SR Ca2+ pump as derived from X-ray crystallography and
sequence studies.
Left bottom: The primary structure of the Na+/K+ pump with the suggested domain breakdown.
To the protein Explorer images of the Ca2+ pump:
The secondary structure and general three-dimensional shape of the SR Ca2+ pump, which were
used for the generation of the animation. Taken from the Protein Data Base and based on the
work of Toyoshima et al. (Nature, 405:647) and Xu et al. (J. of Molecular Biology, 316:201)
a) E1 conformation, side view
b) E2 conformation, side view
(Images generated with the help of Protein Explorer, by Eric Martz, UMass)
To the movie stills of the Na/K pump:
An image from the animation sequence, representing the Na+/K+ ATPase in the context of the
plasma membrane.
Another image, showing a cutout of the membrane with the Na+/K+ ATPase in the hypothetical
E1 conformation.
The Na+/K+ ATPase in the hypothetical E2 conformation, with bound ATP and Na+ ions about to
be released to the outer surface.
To the Topoisomerase structure and stills:
Left: Secondary structure and overall shape of E. coli Topoisomerase I, used in generating the
model. Data by H. Feinberg et al. (Nat. Struct. Biol. 6:961). Image generated with Protein Explorer
by Eric Matz.
Right: Two views of the 3D model of Topoisomerase that was generated. In the bottom one it is
possible to see the secondary structure information used as base data.
To the Topoisomerase mechanism scheme:
An outline of the mechanism of action of Topoisomerase I. From "DNA Topoisomerases:
Structure, Function, and Mechanism" by James J. Champoux (Annu. Rev. Biochem. 70:369).