Harvard University - Department of Molecular & Cellular Biology

MODULATING THE CATALYTIC ACTIVITY OF A PROTEIN PHOSPHATASE BY EXTERNALLY-IMPOSED FORCE

by Alison Grinthal and Nancy Kleckner

February 19th, 2010


(L to R) Beth Weiner, Nancy Kleckner, Alison Grinthal, and Martin Karplus

There is widespread current interest in the roles of mechanical forces for basic cellular process. Many groups are focusing on effects involving forces generated within the cytoskeleton or membranes. The Kleckner laboratory is interested to understand the nature and roles of mechanical forces within chromosomes.

Prof. Kleckner has previously suggested that viewing chromosomes as mechanical objects may be the key to making sense of their behavior (Kleckner et al., 2004. A mechanical basis for chromosome function. Proc. Natl. Acad. Sci. USA, 101, 12592-12597). For example, redistribution of mechanical stress could provide a built-in communication mechanism for transmitting information within, along and between chromosomes. As part of this notion, mechanical stress might arise internally within chromosomes via changes in chromatin structure, which in turn can be modulated both locally and globally during the cell cycle.

But the question of whether such stresses really are a common currency for subcellular information flow ultimately depends on how the scene looks at an even smaller scale, among the individual proteins whose biochemical activities would need to be keyed to stress within larger entities. We report in our recent PNAS article that a large class of proteins involved in many biochemical processes may in fact behave as versatile elastic objects themselves, and we offer suggestions as to how the world of individual proteins may indeed be tuned in to subcellular forces.

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis (Grinthal et al.,2010. Proc. Natl. Acad. Sci. USA 107, 2467-2472). We used molecular dynamics (MD), a computational technique that provides a detailed picture of a protein's expected motions based on its structure, to investigate the mechanical properties of PR65. PR65 is the scaffolding subunit for phosphatase PP2A and is a prototype HEAT repeat protein. A HEAT repeat consists of two alpha helices linked side by side, with a short loop at the top. In many proteins such repeats line up, via contacts between the helices of adjacent units, to form long curved or spiral shapes.

We found that, despite the fact that different repeat units vary widely in sequence, PR65 behaves like a bulk elastic material: it spontaneously bends and twists, and also responds to external force imposed at its ends, via small uniform adjustments spread out along the entire length of the array. At higher levels of imposed force, the molecule fractures at a single specific site (a “flaw”) and then propagates the resulting local stress relief along the length of the molecule in a manner again characteristic of a homogeneous elastic material. In silico mutations reveal that a flaw can be eliminated or created by alteration of a single amino acid. Most importantly, we further show that PR65 retains these properties in its scaffold role, thereby coordinating types of motions that are expected to modulate the activity of the PP2A phosphatase complex. In particular, it can be predicted that imposition of external force on components bound to either end of the PR65 scaffold will tend to open or close the catalytic interface, thus raising the possibility that activity could be modulated by externally-imposed forces exerted by macromolecular substrates.

One of the many roles of PP2A illustrates the potential for such an effect. PP2A localizes to the centromeres of sister chromatids at a point in the cell cycle when those regions are being pulled to opposite poles, and the activity of the complex at this point is thought to be governed by the resulting “spindle tension”. The predicted movements of PR65 suggest that small changes in tension could be directly read out to change PP2A activity via Angstrom-scale modulations of the catalytic active site, without the necessity of wholesale pulling-apart of protein complexes.

An additional beauty of this class of proteins is that instead of being tailored to one specific conformational shift in one specific setting, their design may enable each protein, as well as other family members and proteins of related composition, to act as mechanosensors for a variety of subcellular force sources and as part of a variety of enzymatic and other complexes. Such proteins may, then, be small-scale elastic sensors that turn pulling and pushing within macromolecular ensembles into a mechanism of communication inside the cell.

This work was carried out in collaboration with Dr. Martin Karplus (Harvard Department of Chemistry and Chemical Biology), the inventor of MD analysis. Future studies, to be carried out in collaboration with Dr. Karplus, Dr. Mara Prentiss (Harvard Department of Physics) and Dr. Lynne Regan (Yale University) will combine MD, AFM, solution biochemistry, single molecule pulling and in vivo functional probing of PP2A activity to further explore these possibilities.

Read more in PNAS

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