Lecture 2 - Domains

Question 1

Hierarchy of protein structure

Answer 1

Secondary structure -> Super-secondary structure -> Motif -> Domain

Question 2

What is a protein domain

Answer 2

A polypeptide chain or part of a polypeptide that can fold independently to form a stable tertiary structure

Question 3

Characteristics of domains

Answer 3

• Formed from secondary structures and structural motifs
• Domains are recognisable units of tertiary structure
• If a domain were expressed independently of the rest of a protein, it would form a stable folded structure.
  • motifs would not fold correctly

Question 4

Can domains be whole proteins?

Answer 4

Yes, such as triosephosphate isomerase

Question 5

Can a domain form part of a protein?

Answer 5

Yes, such as pyruvate kinase

phosphoenolpyruvate to pyruvate in glycolysis

Question 6

Name the three groups of domains

Answer 6

alpha-domains - formed from a-helical motifs
beta-domains - contain anti-parallel B-sheets
a/B domains - Formed from BaB motifs predominantly

Derived from Michael Levitt and Cyrus Chothia

Question 7

a-domains

Answer 7

• More general case of leucine zipper motif
• Amphipathic a-helices made up of heptad repeats

H-P-P-H-P-P-P
H - Hydrophobic
P - Polar

• Forms hydrophobic stripe
• Two stripes align to minimise solvent exposure

Question 8

The coiled coil in a-Domains

Answer 8

3.6 residues per turn in a-helix
Hydrophobic stripes twisted

3.5 residues per tuen in coiled-coil
Hydrophobic stripes align

Question 9

Three helix bundle a-domain

Answer 9

Modification of coiled-coil theme; three intertwined a-helices

Hydrophobic residues between helices

Helices can run parallel (e.g. fibrinogen) or anti-parallel (Hsc20 heat shock cognate protein - a chaperone)

Question 10

Four helix bundle a-domain

Answer 10

• Helices not twisted round each other; more cross-over each other (differs to coiled coil)
• Hydrophobic core - hydrophobic residues ‘buried’ between helices

Question 11

The goblin fold

Answer 11

• Found in large groups of related proteins including myoglobin and hemoglobin
• Helix-loop-helix motifs
• Eight helices wrapped around central core - active site heme
• Helix pairs not adjacent with exception of G and H, form anti-parallel pair

Question 12

Explain the up and down barrel

Answer 12

b-Domains are made up from b-fold motifs; b-strands in anti-parallel

• A rolled up sheet of b-sheet fold motifs.
  Last and first b-strand interact via H-bonds to ‘seal’ the roll
• Can be twisted for distorted

Question 13

The beta-barrel beta-domain

Answer 13

Variation on up and down barrel theme but not a simple ‘roll-up’

Strands 4-6 are flipped riund so order in barrel is 1, 2, 3, 6, 5, 4, 7, 8

Superoxide dismutase is an example

Question 14

Greek key proteins as B-domain

Answer 14

Two beta-fold motifs are folded in a Greek key motif

Proteins are made up of a succession of Greek key type folds

y-crystallin - Found in lenses of our eyes - Responsible for maintaining a smooth gradient of refractive index of light

Question 15

The jelly roll as a B-domain

Answer 15

Formed from a series of Greek key motifs, arranged in a different way

e.g. spherical virus coat proteins, concanavalin A

Four continuous b-strands running anti-parallel to a second four

Question 16

The parallel B-helix

Answer 16

Cylinder/helix of parallel B-sheets

Simplest form contains two sheets e.g. Serratia metalloproteinase - first isolated from the Gram-negative bacterium Serratia

Can also contain three b-sheets e.g. pectate lyase (pectate in cell walls)

Question 17

The a/B barrel

Answer 17

• Barrel of multiple B-a-B fold motifs
• Core of hydrophobic twisted B-strands surrounded by hydrophilic a-helices
• Centre of barrel full of hydrophobic side chains
• Active site at 1 end of barrel formed by loops which connect carboxy ends of B-strands to amino end of a-helices

Question 18

The twisted sheet - a/B domain

Answer 18

Unlike the a/b barrel there are a-helices on both sides of the plane of the b-sheet; cannot form a barrel structure

Instead a twisted b-sheet is found at the core of the domain surrounded on all sides by a-helices

Question 19

a/B domain - The horseshoe fold

Answer 19

Leucine rich motif

X-L-X-X-L-X-X-Z-X-L-X-X-X-X-X-X-X-L-X-X-X-L-X-X-X-X
X- any amino acid
Z- Asparagine or cysteine
L- Leucine

Each motif forms a b-loop-a structure stabilised by the leucine residues in the hydrophobic core which pack against each other

Unlike the a/b barrel one face of each b-strand is exposed to solvent

Question 20

Protein modifications

Answer 20

co- and post-translational modifications in vivo

• Addition of hydrophobic groups for membrane localization
• C-terminal - myristoylation, a C14 saturated acid
  N-terminal glycosylphosphatidylinositol (GPI)
• Addition of cofactors for enhanced enzymatic activity
  Flavin (FMN or FAD) may be covalently attached
  Heme cytochrome attachment via thioether bonds with cysteins
• Modification of amino acids
  Hydroxylation of proline in collagen
  Hypusine formation (on conserved lysine of eIF5A)

Question 21

Dynamic Phosphorylation

Answer 21

• Common protein modification in eukaryotes
• Dynamic phosphorylation is key mechanism of cell signalling

Phosphorylation occurs on serine (90%), threonine (10%), and tyrosine (<1%) residues

10-30% of the human proteome is phosphorylated

Can effect protein structure, activity and localisation

Question 22

N-glycosylation

Answer 22

Glycosylation of Asparagine residues occurs co-translationally in the ER
- sequence motif NX(S/T) where X is not proline

N-glycosylation has important structural and functional roles
- increase hydrophillicity
- modulate protein – protein interactions

Question 23

Ubiquitiylation

Answer 23

Ubiquitin - 8.5kDa protein that covalently attaches to lysine side chains

Polyubiquitination targets proteins for degradation

Dynamic mono-ubiquitinoylation regulates activity, localisation, and protein-protein interaction

Other Ubq-like modifiers are involved in regulation
SUMO (Small Ubq-like Modifier)
NEDD8 (Neural Precursor Developmentally Downregulated 8)