Molecular Interactions

Central Theme: Any biological macromolecule must work together with other molecules to carry out its particular functions in the cell and this depends on the ability of molecules to recognize each other specifically.

Central Question: How do molecules that need to interact find each other in the crowded environment of the cell?

Contents in Brief:

Molecular Recognition: The Thermodynamics of BindingA. Thermodynamics of molecular interactionsB. Drug binding by proteins

Specificity of macromolecular recognitionA. Affinity and specificityB. Protein-protein interactionsC. Recognition of nucleic acids by proteins

AllosteryA. Ultrasensitivity of molecular responsesB. Allostery in hemoglobin

[Molecular Recognition: Drug Binding by Proteins]

1. Introduction to molecular recognition

Molecular recognition events are a particularly important subset of molecular reactions involving the binding of one molecule to another and underlie all of the critical processes in biology.

Noncovalent complexes are held together by ionic, hydrogen-bonding, or hydrophobic interactions. They usually dissociate to an appropriate extent at room temperature, leading to a mixture of unbound and bound molecules at equilibrium. The strength of the molecular interaction can be calculated by measuring the concentration of the free and associated species at equilibrium.

An important class of interactions in biology involves more than one ligand molecule binding to a receptor. When the binding of one of these molecules alters the affinity of the other molecules for the receptor, the interactions are referred to as allosteric.

Molecular recognition in biology involves a trade-off b/w affinity and specificity. High-affinity binding is often achieved by increasing the hydrophobicity of the ligand, while specificity relies on hydrogen-bonding interactions.

2. Thermodynamics of molecular interactions — Key concepts

1) The affinity of a protein for a ligand is characterized by the dissociation constant, KD.

2) The value of the dissociation constant, KD, for a binding interaction is the ligand concentration at which half the receptors are bound to ligand.

3) The dissociation constant is commonly expressed in concentration units b/c we ignore the standard state concentrations (I M).

4) For a simple binding reaction (that is, w/o allostery), the binding isotherm is hyperbolic

5) The Scatchard equation recasts the equation governing the binding isotherm into a linear form.

6) Saturable binding is a hallmark of specific binding interactions.7) The value of the dissociation constant determines the concentration range of

the ligand over which the receptor switches from unbound to bound.8) The dissociation constant for a physiological ligand is usually close to the

natural concentration of the ligand.

3. Most drugs are developed by optimizing the inhibition of protein targets

The development of drugs for a specific disease often begins with the identification of the proteins that are involved in steps critical to disease progression, and then proceeds to the design or discovery of molecules that inhibit the function of these proteins. In the initial steps of this process, the molecules that are first discovered to inhibit the target protein, called lead compounds, usually do not have all of the properties that are desirable in a drug.

An example of a lead compound that has been designed to inhibit a protein is shown in the below. The target protein is a protein kinase. The malfunctioning of one such kinase, known as Abl, causes chronic myelogenous leukemia, a cancer in which WBCs proliferate w/o control. Chemists have developed an inhibitor of Abl known as imatinib (marketed as GleevecTM), which blocks the action of Abl and is an effective treatment for the leukemia.

The Abl kinase cannot function unless it can bind to ATP, and so molecules that bind very tightly to the ATP-binding site of the kinase will block the action of the enzyme. Chemists designed a small organic compound to bind to the ATP-binding site of protein kinases. The compound is designed to form hydrogen bonds w/ the protein in a manner that mimics the adenine group of ATP, and it also mimics some aspects of the planar aromatic nature of the adenine group. The chemists chose to add substituents at three positions on the scaffold, leading to the development of lead compounds that inhibited the Abl kinase.

These lead compounds bound only weakly to the Abl kinase, and they also inhibited many other protein kinases. The chemists then tested a large number of compounds in an iterative series of steps, checking for increased affinity for Abl and decreased cross-reactivity at each step. This process of lead optimization eventually led to imatinib.

4. Signaling molecules are protein targets in cancer drug development

The Abl kinase is a tyrosine kinase that catalyzes the phosphorylation of specific tyrosine residues in proteins that control signal transmission b/w cells. Tyrosine phosphorylation is a key signaling switch in animal cells, and it results in the activation of signaling pathways that control cell growth and differentiation. There are many other forms of cancer that are caused by the aberrant behavior of signaling proteins such as the Abl kinase.

Another example of a protein kinase that is an important target in cancer therapy is the epidermal growth factor (EGF) receptor. EGF is a small protein hormone that conveys messages b/w cells and works by binding to and activating EGF receptors that are displayed by cells that it encounters. Like the Abl kinase, the EGF receptor is a tyrosine kinase.

Tyrosine kinases are normally kept off, and their catalytic activity is released only when external signals received by the cell require them to turn on. Malfunctions in the control of the EGF receptor causes some forms of breast and lung cancers, as well as some brain tumors. Two drugs, trastuzumab (HerceptinTM) and erlotinib (TarcevaTM), are used to treat some of these cancers. Trastuzumab is an antibody (a protein) while erlotinib is a small organic compound. Trastuzumab binds to the extracellular portion of the EGF receptor, whereas erlotinib binds to the intracellular kinase domain.

When erlotinib binds to the kinase domain, it occupies much of the space required for ATP binding. The binding of erlotinib and ATP is therefore mutually

incompatible, and erlotinib is known as a competitive inhibitor of the kinase domain with respect to ATP as is imatinib.

5. Most small-molecule drugs work by displacing a natural ligand for a protein

Two classes of drugs that are effective in the treatment of AIDS block the function of proteins produced by the HIV. The HIV viral genome is made up of RNA, and the target of one class of HIV drugs is the enzyme reverse transcriptase, which coverts viral genetic information from RNA to DNA. The DNA then feeds into the cellular transcriptional machinery, thereby directing the production of proteins that are essential for the replication of the virus. HIV drugs known as nucleotide analogs work by mimicking the structure of nucleotide triphosphates, which results in their binding tightly to reverse transcriptase. The nucleotide analogs are so designed that they cannot be incorporated into a growing DNA chain, and they therefore prevent further replication of the viral genome.

Another critical step in the life cycle of the HIV is the cleavage of large precursor protein molecules into smaller fragments that are the properly functional protein units. This is carried out by HIV protease, which is itself encoded by the viral genome. A second class of HIV drugs, known as protease inhibitors, work by binding to the active site of HIV protease and displacing its normal substrates. This prevents HIV protease from working, and the virus is then unable to produce the basic machinery that is required for its replication.

The high affinity of drugs for their targets usually arises from hydrophobic effects. Proteins such as enzymes and signaling switches contain cavities or invaginations

where small molecules are bound naturally. Such cavities provide opportunities for generating the hydrophobic interactions that can drive drug binding.

6. The binding of drugs to their target proteins often results in conformational changes in the protein

Proteins usually have very high specificity for a particular ligand while ignoring very closely related molecules. In 1894, Emil Fisher proposed the lock-and-key model for how a substrate binds to an enzyme. Fisher viewed the protein as a rigid "lock" and the ligand or substrate as a "key."

The problem is that the lock-and-key model does not take into account the intrinsic flexibility of proteins. An alternative model, termed induced fit, was

introduced by Daniel Koshland in 1958. In this model, the binding site is considered to have some plasticity, allowing it to change somewhat to accommodate binding of the ligand or substrate. In fact, proteins are quite flexible and can undergo conformational changes upon binding of ligands. The inhibitor may bind to a conformation of the protein that is different from the one that binds to the natural ligand. It is difficult to predict such conformational changes.

7. Induce-fit binding occurs through selection by the ligand of one among many preexisting conformations of the protein

Besides the one with the lowest free energy, there are many other conformations that are not too much higher in free energy. Such states will be populated according to equilibrium constants determined by their free energies. Most proteins are only marginally stable, and so the unfolded conformation is always populated to some extent. In addition, there are many folded conformations that differ to varying degrees from the one with the lowest free energy.

Even if the term "induced fit" implies that the conformational change is induced by the binding of the ligand and would not occur in the absence of ligand, in reality the free energy of the induced conformation must not be too high, or the

binding of the ligand would be weakened. Induced-fit binding involves the binding of the ligand to conformations of the protein that are populated, although at a low level. This is referred to as conformational selection by the ligand.

In the interaction of ATP with the EGF receptor, it is found experimentally that ATP binds and dissociates very rapidly. This occurs through movements of the lobes of the kinase domain, opening with respect to each other.

8. Conformational changes in the protein underlie the specificity of imatinib

The structure of the Abl kinase when bound to imatinib is different from that seen when the kinase is bound to its natural substrate, ATP. Imatinib penetrates deeper into the body of the protein kinase than does ATP. This is made possible by a change in the conformation of a centrally located structural element in the protein kinase, known as the activation loop.

All protein kinases look very similar when they turn on, b/c they all catalyze the same chemical reaction and have to satisfy the dictates of chemistry. Despite this similarity, each kinase responds to a unique set of activating signals, and they often look quite different when they are switched off.

9. Conformational changes in the target protein can weaken the affinity of an inhibitor

The simple expression that relates the fractional saturation of the protein, f, to the dissociation constant, KD, and the free ligand concentration, [L], was derived by assuming that the protein partitions b/w only two states:

P + L ≈ P·LIf, instead, the protein exists in two populations, only one of which is competent to bind the ligand, then the equilibrium is:

P* + L ≈ P + L ≈ P·L

If the value of ΔG1° is much greater than the value of RT, then P* is the predominant conformation of the protein in the absence of ligand. Some of the intrinsic binding free energy of the ligand, ΔG2°, then goes towards converting P* to P, which weakens the effective binding free energy.

Dasatinib, another drug that is effective against chronic myelogenous leukemia, is ~350 times more potent an inhibitor of the Abl kinase than imatinib. Dasatinib recognizes the active conformation of the kinase domain, which is likely to be the predominant form in cancer cells. Thus, dasatinib does not have to stabilize a less-populated conformation. The price paid for higher affinity is lower specificity.

10. The strength of noncovalent interactions usually correlates with hydrophobic interactions

The affinity of proteins for their natural ligands is usually related to the physiological concentration of the ligand. B/c ATP is quite abundant in cells ([ATP] = 1 mM), the affinity of protein kinases for ATP is relatively weak. Drugs that are kinase inhibitors, in contrast, bind very tightly.

The ATP-kinase complex forms many hydrogen bonds, but it is a much weaker complex than the imatinib-kinase complex, which is stabilized primarily by hydrophobic interactions.

One of the strongest known noncovalent interactions involving a protein is b/w biotin and avidins. Although biotin forms several hydrogen bonds w/ the protein, it is the nonpolar contacts w/ protein sidechains that are the most important factor in the high affinity of the interaction.

11. Cholesterol-lowering drugs known as statins take advantage of hydrophobic interactions to block their target enzyme

Statins are widely used to reduce cholesterol levels in people who produce too much of this steroid lipid. Statins shut down the activity of HMG-CoA reductase, which catalyzes a key step in the cellular synthesis of cholesterol.

One portion of atorvastatin (LipitorTM) resembles HMG and is bound to the enzyme at the same site as HMG. The rest of the drug molecule bears no resemblance to the natural substrate and consists of several aromatic rings, which make the drug significantly bulkier and more hydrophobic than the CoA portion of HMG-CoA.

Nevertheless, hydrogen bonds are very important b/c they are directional and, as a consequence, they impose structural specificity in molecular interactions. Polar groups in proteins, if arranged relatively rigidly, impose geometrical constraints on the placement of donors and acceptors in the ligands that bind near them. Hydrophobic interactions, on the other hand, are not strongly directional. The predominant constraint is the sequestration of the interacting groups away from water, and the precise interdigitation of the groups is of secondary importance.

12. The apparent affinity of a competitive inhibitor for a protein is reduced by the presence of the natural ligand

A complication arises if the natural ligand for the protein is present in abundance during the determination of the isotherm. When measuring the affinity of a drug for a protein kinase, for example, ATP is present at high concentrations (~1 mM) in the cell and protein kinases are normally saturated with ATP. If ATP is present, we have to account for the fact that the inhibitor has to compete w/ ATP for access to the binding site on the protein.

Not all patients given the drug erlotinib respond equally well to the treatment, and it was discovered that patients who responded more favorably had particular mutations in the EGF receptor, while the others did not. The affinity of erlotinib for EGF receptors in cells was studied by measuring the activity of the receptor in the presence of increasing concentrations of the drug. The activity of the receptor is high when there is no inhibitor present, and it decreases to essentially zero when increasing concentrations of erlotinib are added to the cells.

Much less erlotinib is required to shut down the mutant receptor than the normal one — that is, the drug appears to have higher affinity for the mutant receptor.

The parameter that is readily extracted from the data is the concentration of the inhibitor that corresponds to reduction of the activity of the EGF receptor to half its maximal value. This concentration of the drug is referred to as the IC50 value — that is, the inhibitor concentration for 50% inhibition.

We refer to the dissociation constant for the inhibitor binding to the protein as KI in order to distinguish it from the dissociation constant, KD, for ATP binding to the protein. B/c of the competition w/ ATP, we have to modify the relationship b/w IC50 and the inhibitor dissociation constant, KI, to include contributions from KD (for ATP) and the concentration of ATP, [L], as follows:

KI=( IC50 )( KDKD+ [ L ]

)

If we assume that the concentration of ATP in the cell is ~1 mM, and that the dissociation constant for ATP binding to the kinase domains of both the normal and the mutant EGF receptor is ~10 µM (a typical value for protein kinases), then the equation tells us that the derived values of KI for the normal and mutant receptors are 100 times lower than the IC50 (that is, 0.25 nM and 0.038 nM, respectively).

13. Entropy lost by drug molecules upon binding is regained through the hydrophobic effect and the release of protein-bound water molecules

When a ligand molecule that is tumbling freely in solution binds to a protein, there is a significant loss of entropy. Three translational and three rotational degrees of freedom of the ligand are lost, and this acts as a barrier to binding. In addition, certain internal motions of the ligand, such as rotations about single bonds, may also be frozen out, again an unfavorable contribution to the free-energy change associated w/ ligand binding.

Natural ligands, such as ATP, are often very polar, and such molecules can compensate for the unfavorable binding entropy by forming networks of hydrogen-bonding interactions. But for a molecule such as imatinib, which binds w/ high affinity but makes only four hydrogen bonds to the protein, the hydrophobic effect plays a role in stabilizing the interactions b/w drugs such as imatinib and their targets.

The transfer of methane and propane from water to carbon tetrachloride is strongly favored by an increase to entropy, w/ an overall favorable value for ΔG° of -4.1 and -20.0 KJ·mol-1 for of methane and propane, respectively. The favorable entropy change is due to the hydrophobic effect and can be understood as arising from restrictions on the movement of the water molecules in the presence of nonpolar molecules. In these two cases, it is clear that a drug molecule that is sufficiently hydrophobic can overcome the entropic penalty that arises from the loss of its own motion.

The higher the affinity of a drug for its target, the lower the concentration at

which it could be delivered, thereby avoiding side effects. A ready route towards increasing the strength of the interaction is to make the drug more hydrophobic. Unfortunately, increased hydrophobicity brings w/ it many attendant problems, such as decreased solubility and an increased tendency to adhere to various cellular and extracellular components.

An additional favorable entropy term can also play a role in promoting drug binding if the active site of the protein traps water molecules. The presence of polar groups on the surface of the protein often leads to the relatively tight association of water molecules w/ surface groups on the protein. These bound water molecules have a lower entropy than water molecules in the bulk solution. If the binding of a drug causes the release of some of these water molecules, then a favorable entropy change can end up benefiting the process.

14. Isothermal titration calorimetry allows us to determine the enthalpic and entropic components of the binding free energy

The net enthalpy change upon binding is a balance b/w interactions made w/ water and w/ the protein. The entropy change is also a balance involving water. There are two commonly used methods to obtain the enthalpy and entropy changes for a binding reaction. One of them is known as isothermal titration calorimetry. The second method, called as van't Hoff analysis, is to measure the equilibrium constant as a series of temperatures, and thereby derive the binding enthalpies and entropies.

The binding of various statin drugs to HMG-CoA reductase has been analyzed by isothermal titration calorimetry. The top part of (A) shows the heat released upon each injection of ligand solution into the protein solution. The lower part of (A) gives the amount of heat associated w/ binding (on a molar basis) for each point in the titration.

The statins all bind tightly to their target enzyme, but the balance b/w enthalpy and entropy is different in each case. The entropy of binding is favorable in all cases, despite the loss of the translational and rotational entropy of the drug.