Lecture 10 - LDLR Case Study 2

Question 1

How does LDLR ligand binding differ between ApoB and ApoE?

Answer 1

• ApoB requires LDL-A repeats 2-7, whilst ApoE only requires LDL-A repeat 5
• ApoB is a much larger protein, hence requires more interactions to provide sufficient affinity

Question 2

Summarise how YWTD domain is critical for LDLR ligand release

(3 Points)

Answer 2

• In Extracellular environment (pH7), LDLR adopts open conformation that can bind to ligand, whereas in endosomes (pH5) LDLR adopts a closed conformation that releases ligand
• Histidine residues have pKa = 5.5, therefore at low pH they obtain a +ve
• In Endosome, His586 and His582 of YWTD domain gain a +ve charge and interact with conserved negative charge of LDL-A repeats 4/5, outcompetiting the ligand for binding

Question 3

Describe the PCSK9 in terms of:
(i) What it is
(ii) Function

Answer 3

• Protease Enzyme, however its catalytic activity is not involved in LDLR regulation, as cleaved prodomain occludes the active site
• Serves as Chaperone, directing internalised LDLR into the lysosome for turnover

Question 4

How does PCSK9 Interact with LDLR?

Answer 4

Interacts via the EGF Repeat A, therefore the chaperone is capable of interacting with both ligand-bound and unbound LDLR

Question 5

How do PCSK9 Mutations affect LDLR expression on cell surface?

Answer 5

• Some Mutations (e.g., D374Y) lead to PCSK9 having increased affinity for LDLR, or can lead to self-association, further increasing binding affinity
• Other Mutations (e.g.,R218S) lead to PCSK9 having impaired degradation, therefore higher levels in/outside cell

Question 6

What is IDOL? How does it regulate LDLR?

Answer 6

• Inducible Degrader of LDLR (IDOL) is an E3 Ubiquitin Ligase enzyme, which serves as a -ve regulator of LDLR
• Adds K-63 Linked chain to LDLR, signalling the protein for turnover by lysosome

Question 7

What is the Structure of IDOL?

Answer 7

FERM Domain - contains PTB domain, which recognises LDLR cytoplasmic tail

RING Domain - recruits E2 Ubiquitin Enzyme, allowing ubiquitylation of LDLR
* Also mediates dimerisation of IDOL monomers

Question 8

How is IDOL Regulated?

Answer 8

Autoubiquitylation with K-48 Linked chains, which signal protein for turnover by the proteasome