Evolutionary Analysis of the Lysine-Rich N-terminal Cytoplasmic Domains of the Gastric H+,K+-ATPase and the Na+,K+-ATPase

Abstract

The catalytic α-subunits of both the Na,K-ATPase and the gastric H,K-ATPase possess lysine-rich N-termini which project into the cytoplasm. Due to conflicting experimental results, it is currently unclear whether the N-termini play a role in ion pump function or regulation, and, if they do, by what mechanism. Comparison of the lysine frequencies of the N-termini of both proteins with those of all of their extramembrane domains showed that the N-terminal lysine frequencies are far higher than one would expect simply from exposure to the aqueous solvent. The lysine frequency was found to vary significantly between different vertebrate classes, but this is due predominantly to a change in N-terminal length. As evidenced by a comparison between fish and mammals, an evolutionary trend towards an increase of the length of the N-terminus of the H,K-ATPase on going from an ancestral fish to mammals could be identified. This evolutionary trend supports the hypothesis that the N-terminus is important in ion pump function or regulation. In placental mammals, one of the lysines is replaced by serine (Ser-27), which is a target for protein kinase C. In most other animal species, a lysine occupies this position and hence no protein kinase C target is present. Interaction with protein kinase C is thus not the primary role of the lysine-rich N-terminus. The disordered structure of the N-terminus may, via increased flexibility, facilitate interaction with another binding partner, e.g. the surrounding membrane, or help to stabilise particular enzyme conformations via the increased entropy it produces.

Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

“…10). Both contain high frequencies of lysine residues, i.e., 13.41% for the Na + ,K + -ATPase and 13.40% for the H + ,K + -ATPase [51]. In contrast, the Nterminus of Homo sapiens sarcoplasmic reticulum Ca 2+ -ATPase (SERCA1, fast twitch skeletal muscle isoform, accession number O14983) only possesses 47 residues prior to the start of the first transmembrane sequence.…”

“…Although it still has a relatively high lysine frequency of 8.51 % (calculated using the ProtoParam tool, https://web.expasy.org/protparam/ within the ExPASy server of the Swiss Institute of Bioinformatics [59]), this is significantly lower than that of both the Na + ,K + -and the H + ,K + -ATPases. Although the purpose of the N-terminal lysine residues of the Na + ,K + -and H + ,K + -ATPases is still not certain and requires further research, several of them are conserved across vertebrate species [51], which is indicative of an important role, either in enzyme function or its regulation. It is well known, particularly from research on antimicrobial peptides, that lysine residues promote membrane binding [60][61][62] and investigations using peptide fragments of the Na + ,K + -ATPase N-terminus have confirmed membrane interaction [63].…”

“…A possible explanation is that the higher affinity of the E1 state is due largely to an entropic effect rather than an enthalpic one. Theoretical predictions suggest that the N-terminus of both the Na + ,K + -ATPase and the H + ,K + -ATPase are likely to be disordered [51]. Probably for this reason, the N-terminus of the Na + ,K + -ATPase could not be resolved in any of the published X-ray crystal structures [36,[64][65][66].…”

“…A possible explanation for the significant differences in behaviour between the Ca 2+ -ATPase and both the Na + ,K + -and H + ,K + -ATPases could be that they are related to the proteins' N-termini. Both the Na + ,K + -ATPase and the H + ,K + -ATPase have extended extramembrane lysine-rich N-termini on the cytoplasmic side of membrane [51]. From tryptic digestion patterns [33,49,52,53] and experiments in which the N-terminus was removed either by proteolytic cleavage [29,33] or by mutagenesis [54], it is known that the N-terminus undergoes (± 0.1) μM for the native and the truncated enzymes, respectively.…”

Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases

“…Interestingly, the R domains of P-type ATPases have the characteristics of disordered proteins and are therefore highly variable and flexible. The disordered structure of the R domains is likely to facilitate their regulatory function favoring interaction with binding partners and helping to stabilize particular enzyme conformations [ 4 , 5 ].…”