Lecture 2 Flashcards
Explain how zinc finger domains interact with DNA
Zinc fingers consist of an ? helix and a ? sheet region linked together. The ? helical region rests in the major groove of the DNA with amino acid side chains that interact directly with DNA bases via hydrogen bonds. The identity of these interacting side chain amino acids in the helical region of the domain determines which bases it interacts with.
Give some examples of aromatic amino acids
Tyrosine, proline
Which metal ions bind to their binding domain and have a structural role, give some examples of protein that contain these domains
Zn2+ ions bind to zinc domains. Examples of proteins with these domains include botulinum toxin, zinc fingers and DNA binding proteins
Give an example of protein that contains EF-hand domains and describes its role
Calmodulin kinase – requires Ca2+ binding for its structure and regulation. Calmodulin is formed of a single polypeptide chain and contains N and C-terminal globular domains separated by an extended central helix. Each globular domain contains two high-affinity Ca2+ binding sites. Binding of four Ca2+ ions induces major allosteric changes in calmodulin. The two globular heads rotate relative to eachother enabling calmodulin to bind to target proteins and regulate their activity
Which metal ion binding domains have a regulatory role and give some examples of proteins that contain these domains
Ca2+ binding domains have a regulatory role. These include calmodulin and synaptogenin
Binding of SH2 domains to their phosphotyrosine ligands is involved in the formation of signalling complexes, T or F
T
Leucine zipper domains are protein-protein binding or protein-DNA binding domains, T or F
T
How does SH3 bind its ligand
Via aromatic amino acid stacking
What is the role of SH3 domains
SH3 domains bind to proline-rich motifs (poly-proline regions) and acts as an adaptor domain to link proteins together
Which metal ion(s) bind to catalytic metal ion domains
Fe2+ or Cu2+
Describe the role of zinc finger domains
Zinc finger domains are found in the most frequency classes of transcription factors and are DNA binding domains that recognise 3-base pairs of double-stranded DNA
Phosphatases and kinases used PH domains and have a direct role in lipid signalling, T or F
F – kinases and phospholipases contain PH domains involved in lipid signalling
What does SH3 stand for
Src homology 3 domain
Describe the basic structure of an EF-Hand domain
Consists of two ? helices linked by a short loop region of around 12 amino acids that bind to Ca2+
What are EF Hand domains
Ca2+ binding domains that have a regulatory or structural function
What do SH2 domains bind to
Phosphorylated tyrosine residues
EF-Hand motifs bind cations via 5 oxygen containing amino acid side chains, T or F
T
What do SH3 domains bind to
Proline-rich motifs
What do PH domains bind to
Phosphoinositide lipids
What does SH2 stand for
Src homology 2 domain
What is the role of SH2
Acts as an important phosphotyrosine binding domain that is often involved in signalling mechanisms
What is meant by PH domains and what is their function
Pleckstrin homology (PH) domains are involved in membrane binding with functions in signalling and the anchoring of proteins into the membrane
Explain the different functions of zinc finger domains in different proteins
Zinc fingers perform a structural function in protein-DNA interactions or a catalytic function in proteins such as botulinum toxin
What aspect of the SH2 domain provides specificity
Specificity comes from the interaction between the phosphate of the phosphotyrosine and consists of mainly ionic interactions between the negatively charged PO42- and positively charged amino acids. Some hydrogen bonding also contributes
What is the significance of tandem zinc finger repeats usually found in proteins
Tandem zinc finger repeat domains occur as part of larger DNA binding regions. Proteins with more zinc fingers can recognise longer sequences
How do aromatic amino acids interact with water
They don’t, aromatic amino acids are hydrophobic
What is meant by the minimum consensus sequence for SH3
The smallest region which SH3 will bind to. This is the P-x-x-P motif consisting of a 4 residue sequence with first and last residues being prolines
To which domain does Zn2+ bind in a structural mode
Zinc finger domains
Different sizes and valencies of metal ions are liganded by different numbers of amino acids and have different structural requisites, T or F
T
SH3 recognises proline-rich motifs, how does aromatic stacking account for the interdigitating of tyrosine residues with the target poly-proline domains
Aromatic stacking is the process by which the tyrosines contained within the SH3 domain interdigitate with proline residues within the binding proteins target domain. This is caused by an interaction between the aromatic side chains of tyrosine and proline residues with hydrogen atoms attached to the adjacent aromatic ring. This interaction is a dipole-dipole interaction due to the ?- aromatic rings and the ?+ hydrogen atoms. The aromatic residues (tyrosine) of the SH3 domain are positioned so that they can stack with aromatic (proline) residues contained in the proline-rich motifs
As well as being involved in linking signalling components, what other role does SH3 have
Also as a structural role in maintaining multiprotein complexes
Recall how Zn2+ ions interact with zinc finger domains
Zn2+ ions are coordinated tetrahedrally with the zinc finger domains. This is achieved by interactions with 2 cysteine residues from the ? sheet and 2 histidine residues from the ? helix
Which ions bind to EF Hand domains in a structural or signalling mode
Ca2+ and Mg2+
Describe the insulin signalling pathway from ligand binding to recruitment of a scaffold protein, include reference to specific binding domains that are involved
Initially the insulin hormone binds to its receptor on the cell membrane. The activated receptor then autophosphorylates itself at tyrosine residues. Phosphotyrosine then recruits the insulin receptor substrate-1 (IRS1) protein via its PTB domain. The PH domain of IRS1 then binds to phosphoinositide lipids in the plasma membrane. The activated insulin receptor then also phosphorylates the now bound IRS1 at tyrosine residues. Phosphotyrosine within the IRS1 protein then binds to the SH2 domain of the adapter protein Grb2. Grb2 contains 2 SH3 domains and 1 SH2 domain. Once bound by its SH2 domain to IRS1, one of the SH3 domains binds to a proline-rich region of another protein called Sos. Sos then also binds to phosphoinositides in the plasma membrane via its PH domain. The other SH3 domain of Grb2 binds to a proline-rich sequence contained in a scaffold protein. Once the scaffold protein is bound it too binds several other signalling proteins which relay the signal further
