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Lysine residues in Helix 10 of Apolipoprotein A‐I are Responsible for Self‐association
Author(s) -
Basi Rohin Singh,
Burdick John P.,
Burns Kaitlyn S.,
Weers Paul M. M.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04737
Subject(s) - lysine , circular dichroism , chemistry , mutant , denaturation (fissile materials) , apolipoprotein b , helix (gastropod) , guanidine , biochemistry , protein secondary structure , protein structure , alpha helix , amino acid , crystallography , cholesterol , biology , gene , ecology , nuclear chemistry , snail
Apolipoprotein (apo)A‐I is the major protein component of high‐density lipoproteins (HDL) and plays a role in the transport of cholesterol and phospholipids through the blood stream. ApoA‐I is a 28 kDa protein for which the three‐dimensional structure has not been resolved yet. The protein is proposed to be comprised of two domains, a highly structured NT domain (residues 1–189), and a loosely structured CT domain (residues 190–243). The CT domain initiates lipid binding, is the site of self‐association, and is home to six lysine residues at positions 195, 206, 208 in helix 8 and 226, 238, 239 in helix 10. Mutating these six lysine residues to glutamine(6KQ) resulted in a monomeric protein. This implies that these lysine residues play critical roles in ionic bonding needed for self‐association. To determine the precise role of these lysine residues, six single mutants and two triple mutants were engineered and expressed in Escherichia coli . The recombinant proteins were purified through Ni‐affinity and size exclusion chromatography. The two triple mutants with lysine residues in helix 8 or helix 10 are referred as 3KQH8 and 3KQH10, respectively. Circular dichroism (CD) was conducted to determine if the mutations caused disruption of the secondary structure. Estimated α‐helical content was determined by measuring the ellipticity at 222 nm. This showed that α‐helical content was not affected by the mutations. CD was also used to determine the protein stability of the mutants by determining the resistance to guanidine‐HCl induced denaturation. This showed that the mutant proteins retained their stability. Dimethyl suberimidate crosslinking was performed to estimate the oligomeric state of each variant. This showed that 3KQH10‐apoA‐I was predominantly monomeric while the 3KQH8 mutant appeared similar to the wild type with a high degree of crosslinking. Size‐exclusion FPLC provided more detailed information of the oligomeric state of the mutants. The retention time of 3KQH8‐apoA‐I showed a slight change compared to the wild type, but remained an oligomeric state. However, the 3KQH10‐apoA‐I elution profile was nearly identical to that of the 6KQ mutant, indicating that the lysine residues in helix 10 play a critical role in apoA‐I self‐association of the protein. The single lysine mutations only slightly changed the retention time compared to wild type, except for K226 which showed a modest change. This indicates that no single lysine residue is critical for self‐association, but rather three residues are required. Additional mutagenesis studies were carried out to identify negatively charged partner residues that may form a salt‐bridge with the H10 lysine residues. The elution profile of E223QapoA‐I displayed a similar elution profile as K226Q‐apoA‐I, indicating that this residue may form an ionic bond with lysine 226. These studies suggests that charged residues in H10 form intrahelical salt bridges, which promote self‐association of apoA‐I. Support or Funding Information This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers; SC3GM089564 [Weers]; and 5UL1GM118979, 5TL4GM118980, 5RL5GM118978 [BUILD scholarship Basi].