Ozbek, P. | bioRxiv Science

bioRxiv2022

Epistatic effects between amino acid insertions and substitutions mediate toxin-resistance of vertebrate Na+,K+-ATPases

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino-acid substitutions to the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work established that hystricognath rodents (e.g. chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino-acid insertions in the H1-H2 loop, but their functional significance is not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance of NKA in the two lineages that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla lineage unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse lineage, the insertion works in concert with the substitution Q111R to increase CTS resistance while maintaining wild-type ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino-acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

evolutionary biology↗

bioRxiv2019

Sequence-structure-function relationships in class I MHC: a local frustration perspective

Class I Major Histocompatibility Complex (MHC) binds short antigenic peptides with the help of Peptide Loading Complex (PLC), and presents them to T-cell Receptors (TCRs) of cytotoxic T-cells and Killer-cell Immunglobulin-like Receptors (KIRs) of Natural Killer (NK) cells. With more than 10000 alleles, the Human Leukocyte Antigen (HLA) chain of MHC is the most polymorphic protein in humans. This allelic diversity provides a wide coverage of peptide sequence space, yet does not affect the three-dimensional structure of the complex. Moreover, TCRs mostly interact with pMHC in a common diagonal binding mode, and KIR-pMHC interaction is allele-dependent. With the aim of establishing a framework for understanding the relationships between polymorphism (sequence), structure (conserved fold) and function (protein interactions) of the MHC, we performed here a local frustration analysis on pMHC homology models covering 1436 HLA I alleles. An analysis of local frustration profiles indicated that (1) variations in MHC fold are unlikely due to minimally-frustrated and relatively conserved residues within the HLA peptide-binding groove, (2) high frustration patches on HLA helices are either involved in or near interaction sites of MHC with the TCR, KIR, or Tapasin of the PLC, and (3) peptide ligands mainly stabilize the F-pocket of HLA binding groove. Author SummaryA protein complex called the Major Histocompatibility Complex (MHC) plays a critical role in our fight against pathogens via presentation of antigenic peptides to receptor molecules of our immune system cells. Our knowledge on genetics, structure and protein interactions of MHC revealed that the peptide-binding groove of Human Leukocyte Chain (HLA I) of this complex is highly polymorphic and interacts with different proteins for peptide-binding and presentation over the course of its lifetime. Although the relationship between polymorphism and peptide-binding is well-known, we still lack a proper framework to understand how this polymorphism affects the overall MHC structure and protein interactions. Here, we used computational biophysics methods to generate structural models of 1436 HLA I alleles, and quantified local frustration within the HLA I, which indicates energetic optimization levels of contacts between amino acids. We identified a group of minimally frustrated and conserved positions which may be responsible for the conserved MHC structure, and detected high frustration patches on HLA surface positions taking part in interactions with other immune system proteins. Our results provide a biophysical basis for relationships between sequence, structure, and function of MHC I.

biophysics↗