Lecture 5, protein-protein interactions Flashcards
Affinity
The affinity of a particular molecular interaction refers to it’s strenght.
Can be expressed in Kd -> the lower the higher affinity
Can be expressed in delta G -> the more negative the more stable the complex between a ligand and a protein
Specificity
Refers to the preference of a molecule to bind one particular target relative to all others.
Interactions between biological macromolecules usually have hign affinity
Fibroblast growth factor and its receptor
FGF binding displaces an autoinhibition domain in the receptor, which blocks dimerization in the absence of FGF. The kinase domains that phosphorylate each other, which activates the receptor.
Specificity factor
The ratio of the concentration of ligand bound to the target receptor to the total concentration of ligand bound to all other receptors.
The larger the factor the more specific the binding
Ligand concentration and specificity
- When ligand is present at concentrations greater than Kd target, but less than Kd other, most of the ligand will be associated with the target.
- If ligand concentration equals or exceeds Kd other, then both receptors are occupied with ligand, thus lowering specificity.
Charge interactions and specificity
Polar or charged residues at an interface will only contribute favorably to the free energy of binding if there are complementary functional groups at specific positions on the opposite surface.
Lac repressor
In absence of lactose repressor binds tightly to operator
When lactose is present it binds the repressor, reducing it’s affinity for the operator
Two classes of protein-protein interactions
- The surfaces of two folded protein domains make extensive contact with each other. The residues that interact across this interface may come from several different structural elements within the two proteins.
- Mediated by specialized peptide recognition domains (bijv. SH2 and SH3). These domains recognize short peptide segements in other proteins
SH2 domain
Phosphorylated tyrosine bind to the SH2 domain in an extended conformation. The affinity for phosphorylated tyrosine in not very high, but when tyrosine is not phosphorylated it can’t even be detected.
Sensitivity is depended on target sequence
Increased specificity of combinations of domains
If the interactions between peptide binding domains and their targets are not very specific, combinations of these domains are used in a single protein so that the specificity of the final interaction depends on the specificaties of two or more of the component domains beind satisfied simultaneously.
2 domains at specific distance -> only protein with this specific distance between motifs will be bound.
Hydrophobic interactions at protein-protein interface
Residues at the interfaces between proteins interacts trough the packing of hydrophobic residues aigainst each other.
There is an increase in hydrophobic sidechains at the interface (not to many, or it would stick to anything hydrophobic and no longer be soluable) this increases affinity.
Buried surface area
Protein-protein interfaces are characterized by their buried surface area. This refers to the surface area on the interacting proteins that is accessible to water before the complex is formed, but inaccessible in the complex
Interfacial water molecules
Water molecules at the interfaces between proteins.
precisely located on the protein surface, because they form hydrogen bonds with residues on the protein surface.
No entropy gain but they do increase specificity of interaction.
Growth hormone and its receptor
Growth hormone triggers dimerization of receptor.
Tyrosine kinases that are bound to receptor phosphorlyate the receptor.
This recruits signaling proteins (STAT’s) which are phosphorylated and then dimerized
Protein and DNA/RNA binding
Proteins that bind to DNA or RNA typically do so with surfaces that are enhanched in positive charge.
Protein surfaces in contact with RNA or DNA are enriched in arganine and lysine, and depleted of aspartic acid and glutamic acid. The arganine and lyside sidechans, as well as other polar sidechains make hydrogen bonds to the phosphate group. (makes up 60% of hydrogen bonds between protein and DNA)
Double-stranded RNA-binding domain (dsRB)
Many proteins that bind dsRNA contain a module known as dsRB, it makes contact with both the major and the minor groove.
To make contact with both major and minor, the RNA double helix has to be long enough to present two adjacent faces of the minor groove to the protein.
direct readout / base readout
Recognition of DNA by a protein in which a specific pattern of hydrogen bond donors, acceptors and nonpolar groups on the protein is matched to a complementary pattern displayed on the DNA
Indirect readout / shape readout
When a protein recognizes the ability of a DNA sequence to undergo a particular conformational change
Which amino acid is inserted in minor groove and why?
Arginine sidechains are most frequently inesrted into regions of narrowed minor groove. The sequence specificity is not as high for this as it is for most examples of direct readout, because there are multiple A-T and T-A combinations that give rise to a narrowed minor groove.
Palindromic sequence
A binding site in DNA that contains an inverted repeat (two copies of a sequence motif, with one running backwards).
Dimeric proteins can bind to such sequences with two fold symmetry
Proteins binding DNA as dimers
Many DNA binding proteins occur as dimers and recognize palindromic sequences. Each monomer recognizes the same features in each half of the palindromic sequence, allowing both higher affinity and sequence specificity to be achieved.
Zinc fingers
A class of small structural modules that are stabilized by the coordination of zinc by protein sidechains.
The helix of each finger is inserted in the major groove and sequence specific contacts are made to each succesive base. The spacer between modules is quite short, so there are no bases that are skipped, with the three modules reading out the sequence of nine succesive base pairs. The linker between these molecules is flexible and allows them to adapt to the DNA structure.
Cooperative DNA binding
When the binding of one protein to DNA increases the affinity of another protein for DNA.
Interferon-ß enhancher
The production of interferon-ß is triggered by the binding of multiple proteins to control regions in the DNA known as enhancer elements.
The complex of these proteins with DNA is known as an enhanceosome, and its formation is the first stap in the initiation of transcription
KH domain
A class of RNA-binding molecules, that bind with RNA within a cleft, making contact with both the bases and the phophate backbone. Each KH domain interacts specifically with four nucleotides.
Recognition of the RNA by the KH domain relies on hydrophobic bonds between RNA bases and both the backbone and sidechains of the protein.
RNA recognition motifs (RRM)
Small domains that recognize RNA trough stacking interactions.
Most RRM interact with RNA across a surface formed by a 4-stranded ß-sheet. Two conseved aromatic sidechains stack with the bases of the nucleic acids.
allostery
An allosteric protein or RNA is one in which the activity of the protein or RNA is modulated by interactions that occur at a distance from the active site
Graded respons / linear respons
An output that depends on the input function in a hyperbolic fashion, as in a simple binding equilibrium.
Ultrasensitivity
An ultrasensitive system is one in which the response to an input is sharper than expected from a simple binding equilibrium.
Cooperativity
When the binding of a ligand to a protein is ultrasensitive. As more ligand molecules bind to the protein, the saturation of the protein increases more sharply than would be expected for a normal binding event, as if the molecules cooperate with each other.
Can happen with or without allostery
Map kinase pathway
- MAP kinase kinase kinase (MAPkkk) is first activet trough phosporylation
- MAP kinase kinase (MAPkk) is then phosphorylated by MAPkkk.
- Lastly MAP kinase is phospotylated by MAPkk and it will now phosphorylate a number of cellular proteins
Positive and negative cooperativity
When two or more ligands bind to a protein in such a way that they mutually reinforce each of their binding affinities, it is called positive cooperativity.
If a ligand makes it more difficult for each other to bind, the phenomenon is called negative cooperativity
Functions of hemoglobin
Hemoglobin strasports oxygen form the tissues to the lungs. Four oxygen atoms are bound per hemoglobin tetramer, at each iron-containing heme group. Hemoglobin also transports CO2.
Differences Myoglobin and Hemoglobin
- Myoglobin is found in muscle tissue where it serves as a oxygon reservoir. Hemoglobin moves oxygon from the longs to the tissue.
- Myoglobin is a monomer, with one oxygen binding site, while hemoglobin is a tetramer with four oxygen binding sites.
Sigmoid binding curve
When the binding of ligands to a protein is cooperative the shape of the binding vurve is no longer hyperbolic. instead it resembles a S shape and is called a sigmoid.
Allostary for dimer proteins
A dimeric protein with positive cooperativity switches between two conformations.
T state: all binding sites are in low-affinity conformation
R state: all binding site are in high-affinity conformation.
Hill coefficient
This parameter refelects the steepness of the log-log binding isotherm at the point when the protein is half saturated with ligand. A noncooperative system has a hill coefficient of unity. Systems exhibiting positive and negative cooperativity have Hill coefficients that are greater than and less specific than unity.
How does a protein sense it has bound oxygen in one of its binding sites? (hemoglobin)
The change in heme structure that occurs upon binding oxygen are transmitted to other subunits of the tetramer.
The structure of each subunit changes upon oxygen binding -> position of helix F is changed
Bohr effect
The reduction of the affinity of hemoglobin for oxygen that occurs at lower pH, This phenomenon is important because respiration acidifies venous blood, and the Bohr effect facilitates the release of oxygen from hemoglobin.
