Lecture 2

Question 1

Explain how zinc finger domains interact with DNA

Answer 1

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.

Question 2

Give some examples of aromatic amino acids

Answer 2

Tyrosine, proline

Question 3

Which metal ions bind to their binding domain and have a structural role, give some examples of protein that contain these domains

Answer 3

Zn2+ ions bind to zinc domains. Examples of proteins with these domains include botulinum toxin, zinc fingers and DNA binding proteins

Question 4

Give an example of protein that contains EF-hand domains and describes its role

Answer 4

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

Question 5

Which metal ion binding domains have a regulatory role and give some examples of proteins that contain these domains

Answer 5

Ca2+ binding domains have a regulatory role. These include calmodulin and synaptogenin

Question 6

Binding of SH2 domains to their phosphotyrosine ligands is involved in the formation of signalling complexes, T or F

Answer 6

T

Question 7

Leucine zipper domains are protein-protein binding or protein-DNA binding domains, T or F

Answer 7

T

Question 8

How does SH3 bind its ligand

Answer 8

Via aromatic amino acid stacking

Question 9

What is the role of SH3 domains

Answer 9

SH3 domains bind to proline-rich motifs (poly-proline regions) and acts as an adaptor domain to link proteins together

Question 10

Which metal ion(s) bind to catalytic metal ion domains

Answer 10

Fe2+ or Cu2+

Question 11

Describe the role of zinc finger domains

Answer 11

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

Question 12

Phosphatases and kinases used PH domains and have a direct role in lipid signalling, T or F

Answer 12

F – kinases and phospholipases contain PH domains involved in lipid signalling

Question 13

What does SH3 stand for

Answer 13

Src homology 3 domain

Question 14

Describe the basic structure of an EF-Hand domain

Answer 14

Consists of two ? helices linked by a short loop region of around 12 amino acids that bind to Ca2+

Question 15

What are EF Hand domains

Answer 15

Ca2+ binding domains that have a regulatory or structural function

Question 16

What do SH2 domains bind to

Answer 16

Phosphorylated tyrosine residues

Question 17

EF-Hand motifs bind cations via 5 oxygen containing amino acid side chains, T or F

Answer 17

T

Question 18

What do SH3 domains bind to

Answer 18

Proline-rich motifs

Question 19

What do PH domains bind to

Answer 19

Phosphoinositide lipids

Question 20

What does SH2 stand for

Answer 20

Src homology 2 domain

Question 21

What is the role of SH2

Answer 21

Acts as an important phosphotyrosine binding domain that is often involved in signalling mechanisms

Question 22

What is meant by PH domains and what is their function

Answer 22

Pleckstrin homology (PH) domains are involved in membrane binding with functions in signalling and the anchoring of proteins into the membrane

Question 23

Explain the different functions of zinc finger domains in different proteins

Answer 23

Zinc fingers perform a structural function in protein-DNA interactions or a catalytic function in proteins such as botulinum toxin

Question 24

What aspect of the SH2 domain provides specificity

Answer 24

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

Question 25

What is the significance of tandem zinc finger repeats usually found in proteins

Answer 25

Tandem zinc finger repeat domains occur as part of larger DNA binding regions. Proteins with more zinc fingers can recognise longer sequences

Question 26

How do aromatic amino acids interact with water

Answer 26

They don’t, aromatic amino acids are hydrophobic

Question 27

What is meant by the minimum consensus sequence for SH3

Answer 27

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

Question 28

To which domain does Zn2+ bind in a structural mode

Answer 28

Zinc finger domains

Question 29

Different sizes and valencies of metal ions are liganded by different numbers of amino acids and have different structural requisites, T or F

Answer 29

T

Question 30

SH3 recognises proline-rich motifs, how does aromatic stacking account for the interdigitating of tyrosine residues with the target poly-proline domains

Answer 30

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

Question 31

As well as being involved in linking signalling components, what other role does SH3 have

Answer 31

Also as a structural role in maintaining multiprotein complexes

Question 32

Recall how Zn2+ ions interact with zinc finger domains

Answer 32

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

Question 33

Which ions bind to EF Hand domains in a structural or signalling mode

Answer 33

Ca2+ and Mg2+

Question 34

Describe the insulin signalling pathway from ligand binding to recruitment of a scaffold protein, include reference to specific binding domains that are involved

Answer 34

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