Allosteric Control of Guanylate Kinase Using Molecular Springs

In protein dynamics, allosteric regulation is a mechanism in which chemical interaction at an allosteric site, which is to say a site other than the catalytically active protein site, affects the conformation and subsequent function of the protein. Allosteric control is a fundamental control mechanism in cells, especially with regards to enzymes. We use mechanical forces applied to allosteric sites on proteins to mimic a conformational change typically attributed to chemical reactions. In this way, we control the function of the protein with mechanical forces instead of chemical forces. We have attached a semi-rigid DNA polymer to a kinase protein that performs a simple catalytic reaction. This DNA, which acts as a molecular spring, is attached on opposite sides of the soft protein and exerts an outward force on the protein that results in an “open” and functionally incapacitated conformation. This paper investigates how a DNA-binding agent, ethidium bromide, affects the rigidity of our DNA spring. Previous molecular spring experiments with our DNA-protein chimera recorded inhibition of the protein function by singlestranded (ss) DNA interaction with the protein active site. This inhibitory effect lessens our ability to investigate the relationship between mechanical stress and protein function. This paper also inspects the effect that histones have on removing this functional inhibition. Introduction—Since the advent of Koshland’s induced fit theory (1), understanding the relationship between protein form and function has been of fundamental importance to research scientists. We use mechanical stresses applied to guanylate kinase (GK) to deform the conformation of the protein, which affects its function. We are able to use mechanical stress to deform the geometry of proteins because their physical properties resemble soft condensed matter (2). By controlling the mechanical forces we exert on the protein, and using this method with a protein that performs a relatively simple catalytic reaction, we are able to better understand the complex relationship between form and function, while at the same time developing a method to “probe” molecules using mechanical forces. In the long run, the benefits of being able to, in essence, turn a protein on or off, has numerous applications and is of incalculable biological importance. Allosteric regulation is the regulation of a protein by an effector molecule at an allosteric site—that is, a site other than the catalytically active site. We implement a mechanical analog to traditional allosteric control. Previous experiments that used mechanical forces to change protein conformation and function have shown to be successful (3,4,5). Instead of a natural chemical reaction causing the protein’s conformational change, we use mechanical stress caused by the tension stored in a rigid polymer, in this case double-stranded (ds) DNA. We are able to use the energy stored in this molecular spring because of GK’s geometry. A simplified model of GK structure can be identified as three distinct parts—LID, CORE, and NMP-BD—that move with respect to one another (figure 1); this relative motion characterizes changes in the protein conformation. The three parts form a U-shaped structure with the active site located on the inside of the structure (6). Because GK is a soft macromolecule, it naturally fluctuates between different conformations; the forces we apply simply bias the probability of a particular conformation. We favor the open conformation, attaching the dsDNA to the outside of the protein, on opposite sides of the active site, and the dsDNA acts like a bent spring and exerts an outward force of the GK, which changes the GK’s conformation. This “open” conformation is functionally inactive, contrary to the functionally active “closed” conformation. Because GK has a relatively simple relationship between form and function, we are able to inspect how the application of different mechanical forces affects the conformation of the GK. GK catalyzes the reversible phosphoryl transfer from ATP to GMP, producing ADP and GDP. Activity of GK is associated with large conformational changes, about 1 nm. Substrate binding to the GK results in the conformational changes that move as rigid bodies connected by hinges (6). GMP binding induces a large change that brings the NMP-binding region and LID region closer together. Subsequent ATP binding increases the closure on a smaller scale (6). This is a good example of the induced fit mechanism that was mentioned previously. In previous experiments involving the allosteric mechanical control of GK, it was shown that GMP-binding is not necessary for ATP to bind; however, for the phosphoryl transfer to occur, GMP must certainly be bound (3). In this way, GMP controls the reaction of GK through an induced fit mechanism. Without GMP bound to bring the NMP-binding region and LID region closer together, without GMP changing the conformation of GK, the protein function is nonexistent. We control GK by counteracting the work performed by GMP chemically instigating an induced fit mechanism. As mentioned previously, GMP binding is coupled with a large conformational change that “closes” the U-shaped GK. Similarly, the binding affinity of GMP is drastically reduced when GK is in the open conformation (3). If we are able to control the geometry of GK, which is a soft protein that is structurally deformed by simple thermal agitation (2), and hold the GK in the open configuration, we will be able to directly control the reaction rate enzymatically controlled by the protein. Figure 2: (a) GK in the closed conformation with GMP(green) bound (PDB structure 1EX7). (b) GK in the open conformation (PDB structure 1S4Q) Figure 1: Cartoon of GK structure. The red structures indicate where the outward mechanical forces are applied (Cys mutations). Blue: GMPbinding site. Green: ATP binding site. PDB structure 1S4Q. Image from (3). We use dsDNA to apply mechanical forces to the GK to change its conformation. The dsDNA acts as a molecular spring attached to opposite sides of the GK. Previous experiments with a 60mer dsDNA attached to GK (figure 3) showed two-fold inhibition that resulted from the protein being held in the open configuration by the mechanical force exerted by the DNA. The force exerted by the DNA is calculated by the work required to bend it (7):