Lecture 9 - LDLR Case Study 1

Question 1

Define the 5 Classes of LDLR Mutations

Answer 1

1. Class I - disruption of ER synthesis
2. Class 2 - block ER to Golgi Transport
   * Further Divided into 2A and 2B (2A = More Severe)
   * Often occurs due to protein misfolding
3. Class 3 - LDLR reaches surface, but ligand fails to bind
4. Class 4 - LDLR fails to internalise correctly (due to trafficking defects)
5. Class 5 - Block in Ligand-LDLR dissociation (essential for recycling)
   * Often results in trafficking into lysosome for turnover

Question 2

The LDLR Gene consists of 18 Exons. Describe how these map onto the LDLR protein

Answer 2

• Exon 1 - signal peptide (21aa)
• Exons 2-6 - encode LDL-A repeats
• Exons 7-8 - encode EGF Repeats A/B
• Exons 9-13 - encode YWTD/B-propeller domain
• Exon 14 - encodes EGF Repeat C
• Exon 15 - EC region immediately adjacent to membrane, which is site of O-linked Glycosylation
• Exon 16 - encodes membrane-spanning helix (22aa)
• Exon 17-18 - encodes cytoplasmic tail and 3’UTR

Question 3

Define LDL-A Repeats in terms of:
(i) Number
(ii) Function
(iii) How Structure is Stabilised

(4 Points)

Answer 3

(i) 7 repeats (40aa each)
(ii) Ligand Binding (e.g., ApoE/ApoB)
(iii) Conserved folds are stabilised by:
* 6 Highly conserved Cysteine residues, which form 3 S-S bonds
* Ca2+ coordination (also important for ligand binding)

Question 4

How do LDL-A repeats coordinate the Ca2+ ions?

(3 Points)

Answer 4

1. SDE Motif - highly conserved, forming two of ligands of coordinated Ca2+ ion
2. D/E Residues - serve as ligands of Ca2+ ion via acidic side chains
3. W/G residues - interact with Ca2+ ion via the peptide backbone

Question 5

How do LDL-A Repeats recognise ApoE and ApoB, despite them possessing no significant sequence homology?

(2 Points)

Answer 5

• LDL-A repeat Ca2+ coordination produces distinct surface charge distribution pattern (-ve charge), which is critical to ligand binding
• ApoB/E - do not share conserved sequence, but contain highly conserved (+ve) charged regions

Question 6

How is the EGF Precursor Repeat (EGF repeat) fold stabilised?

Answer 6

• Disulphide Bonds
• Ca2+ coordination (Repeats A/B, not C)

Question 7

How is LDLR Produced? Where is it Localised?

Answer 7

• Synthesised as 120kDa precursor, which undergoes both N-linked Glycosylation (ER) and O-linked Glycosylation (Golgi)
• Transported to the Cell surface, where it is localised to clathrin-coated pits until internalisation occurs

Question 8

How/Why does LDLR initially adopt a compact, globular structure in the ER?

Answer 8

• Random Disulphide Bridges produce the compact globular structure, which protects the protein against aggregation in the ER

Question 9

What Types of Chaperones are required by LDLR for correct folding?

(2 Points)

Answer 9

• General Chaperones (e.g., Calnexin, BIP, PDI)
• Private Chaperones (e.g.,RAP, BocA/mesD)

Question 10

Define RAP in terms of:
(i) What it is
(ii) Function

(3 Points)

Answer 10

(i) Private LDLR Chaperone which binds to -ve charge of LDL-A repeats using highly conserved basic residue (Lysine)
(ii) Prevents LDLR-Ligand binding during processing, which could interfere with correct folding
* some cells that produce LDLR also produce ligand (ApoB/E)

Question 11

What is the function of MesD/BOCA?

Answer 11

• Private LDLR Chaperone required for correct folding of YWTD/B-Propeller domain

Question 12

Explain the Function of the LDLR Cytoplasmic Domain

Answer 12

• Contains a NPXY motif, which is recognised by both Clathrin and Cargo Adaptors to recruit receptor into clathrin-coated vesicles (Receptor-mediated endocytosis)
  *

Question 13

Explain how DAB1/2:
(i) Localise to PM
(ii) Recognise Cytoplasmic Tails

Answer 13

(i) DAB1/2 contains a PIP2 binding site, which is enriched in inner leaflet of the plasma membrane
(ii) DAB1/2 contain a PTB domain, which preferentially recognises dephosphorylated Tyrosine (Y)

Question 14

What are the ARH and DAB1/2 proteins?

Answer 14

Cargo adaptors responsible for internalisation of LDLR receptor