Opening Image: Surface model of riboflavin-carrier protein complex. Riboflavin is shown in CPK colors.
Molecular recognition refers to the noncovalent specific interaction between two or more biological molecules exemplified by substrate-enzyme, ligand-receptor, antigen-antibody, DNA-protein, and many other complexes. In addition to noncovalent interactions discussed in previous tutorials, molecular recognition often involves two specific types of interactions with π-electron systems of aromatic rings: cation-π complexes and π-π stacking. These interactions are of major importance in folded proteins and nucleic acids and will be covered in detail in later lectures. Here let's deal with their role in molecular recognition between a small ligand and a protein that binds that ligand.
Electrostatic Forces in π-π Interactions
Interactions between aromatic groups or π-π interactions are important forces in proteins, nucleic acids, and molecular recognition. When aromatic rings are oriented in certain ways, they develop an interaction known as π stacking between their π-electron ring systems. In proteins, the aromatic-aromatic interaction that occurs most frequently is that between two phenylalanine residues. A popular misconception is that aromatic groups should stack on top of one another in a face-to-face manner. However, face-to-face stacking of two phenylalanine rings is not observed because of repulsion between the π-electron clouds.
The cation-π interaction is a strong and specific noncovalent interaction between a cation and the π face of an aromatic ring, such as a potassium ion interacting with negatively charged π-electron cloud of benzene. The main force behind a cation-π interaction is electrostatic. In proteins, cation-π interactions can occur between the cationic side chains of lysine and arginine on one hand and the aromatic side chains of phenylalanine, tyrosine, or tryptophan on the other. While the major contribution to cation-π interactions is electrostatic in nature, most of the amino acid side chains involved in cation-π interactions simultaneously make additional interactions with their local environment, including hydrogen bonds. Over 6 million cation-π motifs are present in protein structures in the Protein Data Bank (PDB). Cation-π interactions are also quite common at protein-DNA interfaces, where they involve positively charged arginine or lysine side chains and aromatic rings of the nucleic acid bases.
Molecular Recognition of Riboflavin
Riboflavin is an essential cofactor for enzymes which catalyze oxidation of a wide variety of substrates. However, the presence of the large, nonpolar ring system lowers the solubility of riboflavin in aqueous solution and a carrier protein is necessary to store and transport riboflavin to enzymes that require this cofactor. spacefill.
of riboflavin-carrier protein complex. Note that the nonpolar part of riboflavin is buried in the protein.
the binding pocket. The riboflavin is stacked between three parallel aromatic amino acid side chains. The aromatic groups interact directly with the flat aromatic π-system of riboflavin. These are π-π interactions. spacefilling model.
Is the hydrophobic effect involved in formation of the riboflavin-protein complex? Yes. The water molecules comprising the solvent cage around the nonpolar faces of the riboflavin are released to the bulk solvent, along with water molecules inside the nonpolar cavity comprising the binding site for the ligand. However, hydrophobic interactions alone cannot explain the binding of riboflavin to the carrier protein. Specific recognition comes from a cumulative contribution of different types of interactions. In large part, specificity comes from π-π interactions, which in turn require a precise fit (shape complementarity) between riboflavin and the binding pocket.
Recognition of Tetramethylammonium Ion
The tetramethylammonium cation (TMA) is isosteric with neopentane, meaning that it has the same number of atoms and configuration of valency electrons. However, TMA is soluble in water, while neopentane is not.
The hydration shell of TMA is very loosely bound. The charge-dipole interaction is much weaker than that for the inner hydration of K+ because the methyl groups prevent close contact of the water dipole with the positively charged nitrogen. For this reason, TMA is called a "quasi-hydrophobic ion." Nature often uses the trimethyl group as a modification of an amino group in situations where the special characteristics of this quasi-hydrophobic cation are needed.
Let's look at how the tetramethylammonium cation binds to a particular protein.
Surface model of human UDP-glucose epimerase. Only one of two chains is shown. TMA is shown as spacefilling model and colored red.
The oxygen atoms of 505 ordered water molecules are colored according to how tightly they are bound to the protein surface. The most highly ordered waters are shown in blue, less ordered in light blue, then bluegreen, light green, yellow-green, and so on. Note that only are ordered within van der Waals contact of TMA; these four water molecules, which are sandwiched between the ligand and protein, are undoubtedly hydrogen bonded to the protein and each other.
Is the tetramethylammonium cation bound to a carboxylate anion? Recall that carboxylate anions are strongly hydrated. And, of course, the charge-charge interaction between TMA and a carboxylate anion would be weak because of distance between the charge centers. So how does protein hold onto TMA?
Stick model of TMA binding pocket. TMA does not bind to a carboxylate anion. Instead it makes van der Waals contact with the of an aromatic amino acid. This is a cation-π interaction.
of TMA pocket. Three bound waters are shown in dark blue. Note that in addition to a cation-π interaction, two oxygens from the protein have replaced water molecules in the hydration shell of the tetramethylammonium cation. It's the sum of the various weak interactions between ligand and protein that account for stabilization of the complex.
What about the hydrophobic effect? The exposed surface of the aromatic side chain is quite hydrophobic, while the tetramethylammonium cation has its charge encased by a shell of hydrophobic methyl groups. Can we distinguish between the hydrophobic and cation-π interactions experimentally? Yes. An example of how biochemists probe noncovalent interactions is covered in the next section.
A Cation-π Cage for Trimethyllysine
Methylation of Lysine 9 in Histone H3 is recognized by Heterochromatin Protein 1 (HP1), which directs the binding of other proteins to control chromatin structure and gene expression.
Surface model of HP1 (gold) complexed to a fragment (residues 5-10) of Histone H3 (CPK). is trimethyllysine (M3L).
The binding the histone tail containing an unmodified lysine (–NH3+) at position 9 is very weak (Kd > 1 mM). As expected, only the histone tail containing trimethyllysine (–N(Me)3+) at position 9 can fully activate HP1 (Kd = 10 μM). Is the tighter binding due to hydrophobic or cation-π interactions or both?
To address this question, Marcey Waters designed a nonpolar isosteric analog of trimethyllysine which differs from M3L only in the presence of carbon in place of nitrogen in the trimethylammonium group.
"7-Deaza-" refers to the replacement of a nitrogen atom in trimethyllysine at position 7 by a carbon. The mimic is less soluble in water than M3L. Explain. [Hint: color atoms by .]
Stick model of the modified histone tail complexed to HP1. Hydrogens of the histone tail are hidden to simplify the display. Trimethyllysine is abbreviated M3L.
Trimethyllsine-9 in the histone tail was replaced with the mimic and the binding of the peptide to HP1 was studied using NMR. No difference in the conformation of the bound peptide from that of the trimethylated peptide was observed.
trimethyllysine cage. spacefilling model.
Note that the trimethylammonium group fits nicely in a cage comprised of three aromatic rings from the protein. This is an exquisite example of cation-π interactions.
Back to our question regarding how much of the binding energy comes from the "quasi-hydrophobic" interaction of the trimethyllysine with the nonpolar cavity versus cation-π interactions. Binding of the histone tail containing the 7-deaza analog to HP1 is thirty times weaker (Kd = 310 μM) than binding to the trimethyllysine tail (Kd = 10 μM). Since we know that 7-deaza-M3L is less soluble in water than M3L, it's clear that the stronger binding of the histone tail containing trimethyllysine is in fact due to cation-π interaction.