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Fulltext Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism

Guan HH, Yoshimura M, Chuankhayan P, Lin CC, Chen NC, Yang MC, et al.

Sci Rep, 2015 Nov 13;5:16441.
PMID: 26563565 DOI: 10.1038/srep16441

ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.
Fulltext Deciphering critical amino acid residues to modify and enhance the binding affinity of ankyrin scaffold specific to capsid protein of human immunodeficiency virus type 1

Saoin S, Wisitponchai T, Intachai K, Chupradit K, Moonmuang S, Nangola S, et al.

Asian Pac J Allergy Immunol, 2018 06;36(2):126-135.
PMID: 28802032 DOI: 10.12932/AP-280217-0037

BACKGROUND: AnkGAG1D4 is an artificial ankyrin repeat protein which recognizes the capsid protein (CA) of the human immunodeficiency virus type 1 (HIV-1) and exhibits the intracellular antiviral activity on the viral assembly process. Improving the binding affinity of AnkGAG1D4 would potentially enhance the AnkGAG1D4-mediated antiviral activity.
OBJECTIVE: To augment the affinity of AnkGAG1D4 scaffold towards its CA target, through computational predictions and experimental designs.
METHOD: Three dimensional structure of the binary complex formed by AnkGAG1D4 docked to the CA was used as a model for van der Waals (vdW) binding energy calculation. The results generated a simple guideline to select the amino acids for modifications. Following the predictions, modified AnkGAG1D4 proteins were produced and further evaluated for their CA-binding activity, using ELISA-modified method and bio-layer interferometry (BLI).
RESULTS: Tyrosine at position 56 (Y56) in AnkGAG1D4 was experimentally identified as the most critical residue for CA binding. Rational substitutions of this residue diminished the binding affinity. However, vdW calculation preconized to substitute serine for tyrosine at position 45. Remarkably, the affinity for the viral CA was significantly enhanced in AnkGAG1D4-S45Y mutant, with no alteration of the target specificity.
CONCLUSIONS: The S-to-Y mutation at position 45, based on the prediction of interacting amino acids and on vdW binding energy calculation, resulted in a significant enhancement of the affinity of AnkGAG1D4 ankyrin for its CA target. AnkGAG1D4-S45Y mutant represented the starting point for further construction of variants with even higher affinity towards the viral CA, and higher therapeutic potential in the future.

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