Structural Biochemistry/Binding

=Ligand Binding=

General Information
One of the functions of proteins is to bind different molecules together. A ligand is a molecule that is recognized by a protein and is able to bind to the target protein. The site at which the ligand binds to the protein is called the ligand-binding site. The ligand-binding site on the protein is quite flexible, making it easier for the ligand to bind to it. Ligand-binding sites are complementary to the protein to which it binds to. As expected, shape plays a significant role in fitting the ligand to the protein. In addition to that, the charge of the ligand and protein also plays a role.

Similar to the ligand-binding site, an active site is a cavity in the protein surface to which enzymes bind to. The active site is surrounded by amino acids that have the highest affinity to the enzyme that will carry out the reaction. Once again, the shape, charge and polarity of the amino acids affect the binding effects of the enzymes.

There are three models for how an enzymes fits into the active site: the lock-and-key model, the induced fit model, and the transition state model. The lock-and key model assumes that the active site is a perfect fit for the enzyme. This model is a more rigid model that does not allow any modification of the active site or the enzyme. The induced fit model is a derivation of the lock-and-key model which still assumes that an active site is designed specifically for the recognition of one enzyme but both the active site and enzyme are flexible and can slightly modify to create the perfect fit. In the transition state model, the active sites binds to the enzyme in its transition state. This effectively lowers the activation energy needed for the reaction to be carried out.



Note: Above is a phosphate-binding protein

In summary, the properties of proteins that affects the ability of enzymes to bind to it are its flexibility, complementarity, surfaces and non-covalent forces. The flexibility allows an easier fit between binding sites and enzymes. The complementarity and surfaces are important factors that contribute to the specificity of an enzyme to the binding site. It may be assumed that covalent forces are used due to their ability to better bind to the enzyme to its active site. However, the strong binding forces of covalent bonds makes it too difficult for active site to release the enzyme. It must be kept in mind that the enzymes do not bind forever in the active site and as a result non-covalent forces are the best for easy recognition of the substrate and releasing it.

Nature of Binding Sites

1. Generally have a higher than average amount of exposed hydrophobic surface

2. Weak interaction can lead to an easy exchange of partners

3. Displacement of water also drives binding events

Ligand Binding by Repeat Proteins
The unique structures of repeat proteins grant them their functions. Their surface area to volume ratio is much higher than typical globular proteins. This characteristic makes them very well suited in mediating protein–protein interactions and organizing multiple proteins into functional complexes.

A property about repeat proteins is that individual repeats and the positions relative between those proteins are the same despite in which protein they occur. As repeat proteins bind to ligands, there is little to no conformational change. Scientists have compared different repeat protein structures with and without ligands bound using RMSD, or root mean square deviation. Studying β-catenin which contains 12 armadillo repeats, scientists have also found a Robo complex. This complex helps to develop bilateral symmetry in insects and vertebrates.

Repeat proteins also bind extended ligands. These proteins use multiple repeats to create an extended surface area for interaction with those extended ligands. This efficiently creates tight binding. Usually, a repeat protein interacts with a peptide that is extended or with a secondary structure element from the target protein.

The fact that repeat proteins are extended helps different regions of these proteins interacts with different ligands which bring the two into a functional complex. This multi-protein structure happens many ways. For an Hsp organizing protein or HOP, two discrete sets of TPR or tetratrico peptide repeat modules (one binding to Hsp70 and the other Hsp90) carry chaperones together to form a functional complex. HEAT repeats are used to make multi-protein complexes in proteins that function differently from average proteins in their nucleocytoplasmatic transport. HEAT repeats in karyopherin form a superhelix and the external convex surface aids in nucleoporin binding while the inner concave face allows for binding with a regulatory protein Ran-GTP. Protein Phosphatase 2A or PP2A is a heterotrimeric protein that has a scaffold subunit to bind to regulatory and catalytic subunits of different HEAT repeat sets. Different versions of the complex exist so different sets of repeats binding within the HEAT domain are independent. An interesting fact is that SV40 small T antigen interferes PP2A’s function by competing with the regulatory subunit which binds to HEAT.

Usually, when there are multiple repeats, a repeat contributes to a binding interface that has the same structural element. There are exceptions to this, though. A helical bundle is formed from the N-terminal capping armadillo repeat when H2 and H3 is packed in the helical BCL9 (β-catenin).Also, a protein like TPR, Fis1, forms complexes with Mdv1 or Caf4 proteins. The N-terminal α-helix of Fis1 takes up the usual hydrophobic groove found on the concave surface on its TPR domain. An α-helix from the target protein comes into a second hydrophobic groove on the concave face. What is atypical, however, is the interaction with Caf4 and a second α-helix from the target protein binding to the convex part of the TPR domain. Finally, a composite surface used for binding the third protein may interact with a repeat protein. Looking at the CSL-Notch-Mastermind complex, we see that Mastermind interacts with Notch1 and CSL simultaneously but neither of these undergo a big conformational change when the complex forms. This means that Mastermind distinguishes the composite surface from both rather than binding to either through allosteric induction.

Repeat proteins do more than just protein-protein interactions. More and more repeat proteins are found to bind to ligands. Instead of specialized folds, the same repeat and fold are binding to many different types of ligands. A well-known example is the toll-like receptor or TLR from mammalian immune systems that bind proteins, lipoproteins/peptides, and nucleic acids. HEAT repeats can also bind in many ways. They are usually found intervening protein-protein interactions but can be found binding nucleic acids.

Protein designers are working on making new repeat proteins because simple and short repeat proteins can be used to bind many ligands using scaffolds. Many sequence alignments and structural characterizations allow for a clear description of structural and functional residues that are important. Two complementary strategies are being used: 1- introducing novel binding specificities onto existing repeat scaffolds and 2 – creating new scaffolds onto which known binding sites are grafted.

Organization of Multi-protein Complexes
The extended, modular nature of repeat proteins allows for different sections of the protein to be used to bind many different ligands and then bring them together to form functional complexes. An example of this function of repeat proteins is the Hsp organizing protein(HOP), in which two defined sets of TPR modules each bind to Hsp 70 and Hsp 90 to bring them together into a complex.

Typically, when binding involves multiple repeats, each repeat contributes to the binding interface with the same structural element. In a given repeat protein, its binding interface could be formed by only H1 helices, or antiparallel beta strands, etc.

Research
Repeat proteins have become key targets for protein design. Two strategies have been employed in synthesizing new repeat proteins: 1) addition of new binding specificities onto existing repeat scaffolds, and 2) synthesize new scaffolds onto which known binding sites are inserted. For example, Ank repeats have been used extensively in the first strategy presented.In another example, a TPR module has been designed by grafting Hsp90-binding residues onto a synthesized consensus TPR scaffold. What this has done is create a new protein that has greater affinity and specificity for Hsp90 than natural Hsp90 co-chaperones. This has had significant impact in fighting breast cancer. Synthesizing stronger versions of existing repeat proteins is one way in which the second strategy is used.