Red blood cell invasion by Plasmodium vivax: Structural basis for DBP Engagement of DARC

Question 1

Detail the significance of this study?

Answer 1

This study was significant in that it achieved the following:

1) NMR was used to solve the structure of DBP-RII bound to the core region of DARC
2) It was determined that binding occures in a multistep fashion
3) It was determined that point mutations in those DBP-RII residues that contact DARC result in a loss of RBC binding

Ultimately, this work will allow for the development of models that examine inter-species infection barriers, immune evasion mechanisms, receptor-ligand specificity in P. knowlesi, and what mechanisms underlie P. vivax immunity. This will all lead to the development of better treatment options for malaria.

p. 1 Abstract

Question 2

As detailed in the paper, describe the chemical interactions that occur for entry of Plasmodium vivax.

Answer 2

In order to invade RBC’s, P. vivax must release the special contents of apical organelles (micronemes and rhoptries). DBP, one of the contents of these organelles, is a member of the EBL family of proteins. They use DBL domains to bind to specific RBC receptors with high affinity.

p.1 Introduction

Question 3

How was binding to the central region of DARC shown?

Answer 3

Using NMR, it was shown that DBP-RII binds to the central region of DARC. Specifically, it was shown that peaks that corresponding to the central region of DARC 1-60 became broadened and shifted or disappeared in the bound complex.

These results indicated that residues in the central region of the DARC ectodomain are perturbed upon interaction with DBP-RII and are most likely to make direct contacts with one another to form the binding domain.

p. 2

Question 4

How was it shown that the N-terminal region or the C-terminal region of the DARC is not necessary for interaction with DBP-RII?

Answer 4

Using NMR comparisons of the bound and unbound states of DARC, it was shown that there was only modest broadening in the first 15-16 aa residues in the N-terminus region. This suggests that the region remains unstructured upon binding and does not dirrectly contact DPB-RII.

In the C-terminus, aa residues from 11-60 display some line broadening but the region is still fairly unperturbed upon binding to DBP-RII.

p. 3 results

Question 5

How was Isothermal Titration Calorimetry (ITC) used to confirm the stepwise formation of the heterotetramer of DBP:DARC?

Answer 5

Isothermal titration calorimetry (ITC) is a physical technique used to determine the thermodynamic parameters of interactions in solution. It is most often used to study the binding of molecules.

ITC was used in this study to examine the mechanism of DBP:DARC binding by titrating DARC into DBP-RII.