TLR4 is a receptor for bacterial lipopolysaccharides (LPS) that cause septic sho
ID: 70519 • Letter: T
Question
TLR4 is a receptor for bacterial lipopolysaccharides (LPS) that cause septic shock. The crystal structure of TLR4 (blue and green) bound to MD-2 (gray) and lipid A (red in Figure a and b, and structural in Figure c) are shown in the figures below. Please answer the following questions based on these structures: What are the dominant secondary structures of TLRR4 and MD-2? Please show in the Ramachandran plot the region you will find most of the amino acid residues of TLR4 and MD2, respectively. A close up of MD-2 is shown below. What is the quaternary structure of the TLR4/MD-2/lipid-A complex? What kind of non-convalent interactions mediate the biding of lipid-A to MD-2? What kind of residues are likely involved lipid A and by MD-2? Please give five example of these amino acids. TLR4 has 600 residues and MD-2 has 150 residues. If we assume this complex as shown in the structures are stable, what is the molecular mass of the complex you expect by gel filtration chromatography? What will you see on a SDS-PAGE gel for this complex in presence of DTT?Explanation / Answer
a) TLR4 secondary structure
The crystal structure of TLR4 investigated in mouse was referred to as the member of the LRR superfamily. Usually LRR subfamily proteins are horse shoe shaped. Their concave surface is made of parallel beta strands and the convex surface is made of 310 helices and loops. Unique twist angles and radii are observed in the parallel beta sheets of the entire TLR4 protein. Beta sheets of this protein are known to possess N-terminal, C-terminal and central domains. N-terminal domain spans from 26th amino acid to 201th amino acid. The twist angle and radius of this domain resembles well with that of the typical LRR (leucine rich repeat) family proteins. The radius of the beta sheet of the C-terminal domain is 28 percent bigger than that of the N-terminal domain. Central domain consists of 35 percent smaller radius and three times larger twist angle than that of TLR3. The LRRNT, LRR and LRRCT modules span from 1 to 6, 7 to 12 and from 13 to 22 respectively. LRR module consists of two variable amino acids in between the first and second leucines that are conserved. The typical conserved asparagine ladder in the LRR modules of the subfamily is absent in TLR4 from LRR modules 9 to 12. The LRR module length of the central domain varies and ranges from 20 AA to 30 AA residues. The LRR module length is associated with the entire shape of the horse-shoe like structure. LRR modules are conserved in all the six subfamilies of the LRR superfamily. Short LRR modules consist of loops in the convex region and the longer modules comprise of alpha helices.
Secondary structure of MD-2
MD-2 protein has beta cup fold structure comprising of two antiparallel beta sheets. These sheets consist of three and six beta strands. This type of folding in MD-2 has the beta sheets separated towards one side of the protein with a hydrophobic internal core exposed for ligand binding. The separation forms a large internal pocket. This pocket is big enough to give space for lipopolysaccharides. The internal surface of the pocket has hydrophobic residues while the opening region consists of positively charged residues that help in the binding of lipopolysaccharides.
b) Dihedral angles in Ramachandran plot for the aminoacids in TLR4
Alpha = 90 degrees
Beta = 115.72 degrees
Gamma = 90 degrees
Dihedral angles in Ramachandran plot for the aminoacids in MD-2
Alpha = 90 degrees
Beta = 90 degrees
Gamma = 90 degrees
99.5 percent of all AA residues in the TLR4-MD2-lipid complex investigated in mouse, fall in the allowed regions.
c) The protein complex in mouse consists of A, B, C and D polypeptide chains.
d) The large hydrophobic cavity of MD-2 binds directly with the lipid A which is the active center of LPS.
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