Protein-Ligand Interactions (Biochemistry) PDF

Protein-Ligand InteractionsIntroductionBiochemistry places significant emphasis on enzymes, which facilitate the chemical reactions needed to sustain life. This important class of protein is covered extensively in Unit 2. However, many other proteins fulfill important biological roles that do not in...

Protein-Ligand InteractionsIntroductionBiochemistry places significant emphasis on enzymes, which facilitate the chemical reactions needed to sustain life. This important class of protein is covered extensively in Unit 2. However, many other proteins fulfill important biological roles that do not involve chemical reactions. For instance, many proteins help to transport molecules from one location to another within an organism. Others help to store molecules for later use, while still others receive chemical signals from the environment and transmit them to the cell, causing a biological response.Many nonenzymatic proteins carry out their functions by binding to one or more specific molecules. Any molecule that specifically binds to a protein is called a ligand for that protein. This lesson discusses fundamental aspects of protein-ligand interactions.3.1.01 Ligand BindingProteins bind their ligands through. These interactions form between the ligand and amino acid residues or in a region of the protein called the binding site or binding pocket. The chemical groups in a binding site have a specific orientation and, as a result, often bind with specificity (ie, they preferentially bind ligands with specific structural features). Some proteins are highly specific and bind only one compound or a few structurally similar compounds. Other proteins are less specific and bind a more diverse set of compounds that share a few key features.Mutations within the binding pocket of a protein may significantly alter function, particularly for highly specific protein-ligand interactions. In some cases, even may reduce binding. For instance, and have nearly identical structures, but the aspartate side chain is shorter than that of glutamate. Therefore, mutating glutamate to aspartate (or vice versa) in a binding pocket may impact binding because the angles required for interactions with the ligand change. Figure 3.1 shows an example of the effects of such a mutation. Chapter 3: Nonenzymatic Protein Activity90Figure 3.1 Mutations in binding pocket residues can significantly affect binding strength.Ligands may range in size from small molecules or ions (eg, O2 binding to hemoglobin) to large molecules (eg, DNA binding to a ). Frequently, the ligand for one protein is another protein, leading to the formation of a protein-protein complex. This arrangement arises through noncovalent interactions between amino acid residues. In this way, protein complexes are similar to but differ in that each unit in a protein complex can function independently, whereas the individual subunits in a protein with quaternary structure function only when bound together.Antibodies illustrate several aspects of quaternary structure and protein complex formation. An antibody is a protein that consists of four polypeptides---two heavy chains and two light chains---linked together through various noncovalent interactions and disulfide bonds. Without these interactions, an individual chain cannot bind its ligand and the antibody is not fully functional. Because these interactions are required for the structure and function of the antibody, they constitute quaternary structure. Chapter 3: Nonenzymatic Protein Activity91The ligand for an antibody is called an antigen. Antigens are often proteins found on the surface of an infectious organism. The specific portion of the antigen to which the antibody binds is called an epitope, which may include such as carbohydrates or phosphate groups. The portion of the antibody that binds the epitope is called the paratope. The antibody and the antigen are both functional regardless of whether they are bound to each other, so their binding interactions constitute protein-protein complex formation rather than quaternary structure.In some cases, a complex may consist of multiple proteins bound to each other. For instance, an antibody may bind its antigen through the paratope (ie, the binding site) while simultaneously binding a cell receptor protein at another site. The cell receptor protein may in turn be linked to many additional proteins. Figure 3.2 depicts an antibody binding both an antigen and a receptor protein.Figure 3.2 Interactions between an antibody and other proteins.Ligand binding generally causes the protein to undergo a conformational change that results in a biological response. Many drugs are designed to interact with a binding pocket without causing the biological response. For instance, cancer proliferation often involves overactivity of the transcription factor Myc, which must bind another protein called Max before binding to DNA to induce transcription. Various drugs that disrupt Myc-Max interactions (Figure 3.3) have been tested for anticancer properties. Chapter 3: Nonenzymatic Protein Activity92Figure 3.3 A protein-drug interaction disrupting the biological function of a protein.3.1.02 The Dissociation Constant KdDifferent proteins bind their ligands with different strengths, and a given protein may bind several different ligands, each with a different strength. The overall strength with which a protein binds a ligand is called its affinity for that ligand. Strong protein-ligand interactions are high affinity, and weak interactions are low affinity. Typically, the affinity of a protein for its ligand is measured by the dissociation constant Kd. Kd is the for the process in which the protein-ligand complex (PL) dissociates into free protein (P) and free ligand (L).PL ⇌P+LAs with all equilibrium constants, a large value indicates that products are favored whereas a smaller value indicates greater reactant favorability. Because Kd uses PL as the reactant, a small Kd value indicates that PL formation is favored (ie, dissociation is not favored). Therefore, small Kd values correspond to tight (ie, strong) binding and high affinity.Concept Check 3.1The spike protein on the original variant of the SARS-CoV-2 virus contains a glutamate residue at position 484. In later variants, this residue mutated to lysine, reducing the binding strength of antibodies that target an epitope containing this residue. Based on this information, how does this E484K mutation affect the Kd of the interaction between these antibodies and the spike protein? Chapter 3: Nonenzymatic Protein Activity93By definition, an equilibrium constant is the equilibrium concentration ratio of all products to all reactants, with each concentration raised to the power of its. Therefore, Kd is defined as𝐾d=\[P\]eq\[L\]eq\[PL\]eqEach concentration has units of. Because the numerator contains two concentrations multiplied by each other and the denominator contains only one concentration, the units do not fully cancel, and Kd also has units of molarity. However, because Kd is usually small, it is often to units of mM, μM, or nM. An example Kd calculation is shown in Figure 3.4.Figure 3.4 Example of a Kd calculation given known concentrations of PL, P, and L at equilibrium.Protein binding typically behaves as a reversible single-step process---either the protein binds its ligand or the protein-ligand complex dissociates---but no intermediates form in either case. Therefore, the rate of complex formation is given asrate=𝑘on\[P\]\[L\]and the rate of complex dissociation israte=𝑘off\[PL\]in which kon and koff are the binding and unbinding , respectively. At equilibrium, binding and unbinding occur at the same rate, and therefore𝑘off\[PL\]eq=𝑘on\[P\]eq\[L\]eqRearranging this equation yields𝑘off𝑘on=\[P\]eq\[L\]eq\[PL\]eq=𝐾d Chapter 3: Nonenzymatic Protein Activity94A small Kd indicates a relatively small (ie, slow) unbinding rate constant or a large (ie, fast) binding rate constant (or both), confirming that a small Kd indicates strong binding affinity. Conversely, a large Kd indicates a relatively large (ie, fast) unbinding rate constant or a small (ie, slow) binding rate constant (or both), confirming that a large Kd indicates weak binding affinity. Note, however, that if only Kd is known, the specific values of the rate constants cannot be determined because various combinations of kon and koff could produce the same Kd (eg, 2/1 = 2, 4/2 = 2).Concept Check 3.2A protein binds its ligand with a rate constant kbinding of 3.5 × 104 M−1s−1 and unbinds with a rate constant kunbinding of 1.4 × 10−1 s−1. What is the dissociation constant Kd for this process?For any chemical process, the equilibrium constant is related to the standard free energy change ∆G° of that process. In the case of protein-ligand dissociation, the relationship is given by the equation ∆𝐺°=―𝑅𝑇 ln (𝐾d)in which R is the universal and T is of the system. Therefore, if the standard free energy change of dissociation at a given temperature is known, the Kd value can be calculated. More generally, any Kd smaller than 1 M corresponds to a positive (ie, unfavorable) ∆G° of dissociation. Most biochemically relevant Kd values are well below 1 M, and protein-ligand dissociation is usually quite energetically unfavorable. Because the ∆G° of binding has the same magnitude as that of dissociation but the opposite sign, this also means that binding is favorable.Concept Check 3.3The following table shows Kd values for a protein-ligand interaction at various temperatures.Temperature (°C)Kd (M)303.1 × 10−4354.8 × 10−4402.3 × 10−3Which temperature corresponds to the most negative value for ∆G° of binding? (Assume any changes in the −RT portion of the equation have a negligible impact on ∆G° over the range of temperatures used.) Chapter 3: Nonenzymatic Protein Activity953.1.03 Binding CurvesThe Kd for a protein-ligand interaction can be determined by generating a binding curve. These curves are produced by gradually adding ligand to a known amount of protein and measuring the response. The results are plotted as the fraction of total protein bound (θ) on the y-axis against the concentration of ligand added on the x-axis.For a single ligand binding to a single pocket, the binding curve fits the equation𝜃=\[L\]𝐾d+\[L\]When plotted, this equation yields a hyperbolic curve that rises quickly, then levels off as saturation occurs (ie, when nearly all proteins in solution are bound). When \[L\] = Kd, a substitution can be made so that the equation becomes𝜃=𝐾d𝐾d+𝐾d=𝐾d2𝐾d=12In other words, for single protein--single ligand interactions, Kd equals the concentration of free ligand at which 50% of the proteins in solution are bound and 50% are unbound (see Figure 3.5).Figure 3.5 Example of a hyperbolic binding curve with Kd marked.This is consistent with the fact that a lower Kd corresponds to increased binding affinity. A smaller Kd means a smaller amount of ligand is required to bind half the proteins in solution.For gaseous ligands (eg, O2), is easy to measure and is often used instead of concentration. The partial pressure at which 50% of proteins are bound (ie, the P50) is used instead of Kd in this case, and the binding equation becomes𝜃=𝑃gas𝑃50+𝑃gas Chapter 3: Nonenzymatic Protein Activity96The curve generated by this equation looks identical to binding curves that use ligand concentration, except that the x-axis has units of pressure (eg, atm, mm Hg).Concept Check 3.4The following graph shows the binding curve for interactions between myoglobin and oxygen under physiological conditions.Based on the graph, what is the approximate P50 of the myoglobin-oxygen interaction?Alterations to a protein that change its affinity for its ligand (eg, post-translational modifications) cause changes in the appearance of the binding curve. For example, conditions that increase the affinity cause the Kd of the protein-ligand interaction to decrease. On a binding curve, Kd corresponds to a position on the x-axis, and in this case, the position of Kd on the x-axis moves to the left (ie, the curve is left-shifted). In contrast, conditions that decrease affinity cause an increase in Kd and a right shift on a binding curve. Shifts to the left and right are shown in Figure 3.6. Chapter 3: Nonenzymatic Protein Activity97Figure 3.6 Left and right shifts in response to conditions that increase and decrease affinity, respectively.3.1.04 Allostery and CooperativityBinding proteins usually have a primary ligand, which is the ligand that is required for the protein to perform its biological function. This ligand binds to the protein at the binding pocket. However, many proteins can bind ligands other than their primary ligand at a site other than the primary binding pocket. Sites on a protein that bind ligands other than the primary ligand are called allosteric sites (see Figure 3.7).These other ligands, called allosteric effectors, cause proteins to undergo conformational changes that can affect the whole protein. Even small changes in conformation may realign the functional groups in the binding pocket and alter the affinity for the primary ligand. Some allosteric effectors increase a protein\'s affinity for its ligand while others decrease affinity. In this way allosteric effectors can regulate protein-ligand binding by helping proteins to bind and release their primary ligands as needed. Chapter 3: Nonenzymatic Protein Activity98Figure 3.7 An allosteric site and effector compared to the binding pocket and primary ligand.Concept Check 3.5A scientist proposes that a specific compound is an allosteric effector for protein P. The scientist further hypothesizes that the compound increases the affinity of P for its ligand L. If the scientist is correct, how will the binding curve of the interaction between P and L change when the compound is added?One important consequence of allosteric interactions is the phenomenon of binding cooperativity. Broadly, cooperativity is any biochemical process that alters the ability of another process to occur. For instance, protein folding and unfolding are cooperative because formation of secondary structures increases the ability of tertiary structure to form. With respect to binding (and unbinding), cooperativity refers to the effect of one binding interaction on other binding interactions by the same protein.Cooperativity may be positive or negative. occurs when binding interactions at one site increase the affinity of other sites for their ligands, and negative cooperativity refers to binding interactions that decrease the affinity of other binding sites. Thus, allosteric effectors that increase affinity (ie, decrease Kd) induce positive cooperativity, and those that decrease affinity (ie, increase Kd) induce negative cooperativity.An especially important form of cooperativity occurs when multiple, identical ligands bind to multiple binding pockets on the same protein. This type of cooperative binding often occurs in proteins with quaternary structure, in which each subunit contains one binding pocket. When one pocket binds its ligand, the other binding pockets may change shape and therefore may change affinity for the ligand. This form of cooperativity is depicted in Figure 3.8. Chapter 3: Nonenzymatic Protein Activity99Figure 3.8 Schematic of positive cooperativity in a protein with two pockets that bind the same ligand.The extent to which cooperativity occurs in proteins with multiple binding sites for the same ligand is measured by the Hill coefficient n. A Hill coefficient above 1 indicates positive cooperativity, a Hill coefficient of exactly 1 indicates that no cooperativity occurs, and a Hill coefficient below 1 (but greater than 0) indicates negative cooperativity.For a protein with x identical binding sites, the Hill coefficient can be no greater than x and no smaller than 1/x, but it may be anywhere in between. For example, hemoglobin has four oxygen binding sites and a Hill coefficient of 2.8.The maximum possible Hill coefficient for a protein (eg, 4 in the case of hemoglobin) indicates perfect cooperativity---that is, cooperativity such that either all binding sites bind their ligand simultaneously or none bind at all. In practice, proteins are rarely perfectly cooperative. Table 3.1 summarizes the meanings of various Hill coefficients for a protein with four subunits. Chapter 3: Nonenzymatic Protein Activity100Table 3.1 Interpretation of the Hill coefficient for a protein with four binding sites.When a protein has a Hill coefficient n other than 1, the shape of the binding curve changes. In positive cooperativity (ie, n \> 1), increases in ligand concentration initially cause small changes in binding levels because the affinity is low. This results in a shallow initial slope on the binding curve.However, as ligand concentration reaches a point where most proteins in solution have one binding site filled, the other binding sites gain higher affinity for the ligand. At this point, small increases in ligand concentration cause a greater increase in the percentage of binding sites that are filled, and the slope of the curve increases dramatically. Once all binding sites are filled, the curve levels off. Consequently, positive cooperativity yields a sigmoidal (ie, S-shaped) curve (see Figure 3.9).Negative cooperativity (n \< 1) has the opposite effect. Initially, increases in ligand concentration induce a larger increase in binding than occurs at higher ligand concentrations. As binding continues, other binding sites decrease in affinity for the ligand, so additional ligand causes progressively smaller increases in binding. This yields a curve that looks similar to noncooperative binding, but the curve flattens much more quickly. Because negative cooperativity yields a similar shape to no cooperativity, identification of negative cooperativity from a graph is unlikely on the exam. Chapter 3: Nonenzymatic Protein Activity101Figure 3.9 Example graphs of protein-ligand binding with positive, negative, and no cooperativity.Any protein that exhibits binding cooperativity also exhibits unbinding cooperativity. For example, if a protein with positive cooperativity has all sites bound, then removing one ligand makes it easier to remove additional ligands. This can be represented on an unbinding curve as an inverted sigmoid as shown in Figure 3.10.Figure 3.10 Example of a ligand unbinding curve exhibiting positive cooperativity.