4 research outputs found
Cardiolipin occupancy profiles of YidC paralogs reveal the significance of respective TM2 helix residues in determining paralog-specific phenotypes
YidC belongs to an evolutionarily conserved family of insertases, YidC/Oxa1/Alb3, in bacteria, mitochondria, and chloroplasts, respectively. Unlike Gram-negative bacteria, Gram-positives including Streptococcus mutans harbor two paralogs of YidC. The mechanism for paralog-specific phenotypes of bacterial YidC1 versus YidC2 has been partially attributed to the differences in their cytoplasmic domains. However, we previously identified a W138R gain-of-function mutation in the YidC1 transmembrane helix 2. YidC1W138R mostly phenocopied YidC2, yet the mechanism remained unknown. Primary sequence comparison of streptococcal YidCs led us to identify and mutate the YidC1W138 analog, YidC2S152 to W/A, which resulted in a loss of YidC2- and acquisition of YidC1-like phenotype. The predicted lipid-facing side chains of YidC1W138/YidC2S152 led us to propose a role for membrane phospholipids in specific-residue dependent phenotypes of S. mutans YidC paralogs. Cardiolipin (CL), a prevalent phospholipid in the S. mutans cytoplasmic membrane during acid stress, is encoded by a single gene, cls. We show a concerted mechanism for cardiolipin and YidC2 under acid stress based on similarly increased promoter activities and similar elimination phenotypes. Using coarse grain molecular dynamics simulations with the Martini2.2 Forcefield, YidC1 and YidC2 wild-type and mutant interactions with CL were assessed in silico. We observed substantially increased CL interaction in dimeric versus monomeric proteins, and variable CL occupancy in YidC1 and YidC2 mutant constructs that mimicked characteristics of the other wild-type paralog. Hence, paralog-specific amino acid- CL interactions contribute to YidC1 and YidC2-associated phenotypes that can be exchanged by point mutation at positions 138 or 152, respectively
DataSheet1_Cardiolipin occupancy profiles of YidC paralogs reveal the significance of respective TM2 helix residues in determining paralog-specific phenotypes.PDF
YidC belongs to an evolutionarily conserved family of insertases, YidC/Oxa1/Alb3, in bacteria, mitochondria, and chloroplasts, respectively. Unlike Gram-negative bacteria, Gram-positives including Streptococcus mutans harbor two paralogs of YidC. The mechanism for paralog-specific phenotypes of bacterial YidC1 versus YidC2 has been partially attributed to the differences in their cytoplasmic domains. However, we previously identified a W138R gain-of-function mutation in the YidC1 transmembrane helix 2. YidC1W138R mostly phenocopied YidC2, yet the mechanism remained unknown. Primary sequence comparison of streptococcal YidCs led us to identify and mutate the YidC1W138 analog, YidC2S152 to W/A, which resulted in a loss of YidC2- and acquisition of YidC1-like phenotype. The predicted lipid-facing side chains of YidC1W138/YidC2S152 led us to propose a role for membrane phospholipids in specific-residue dependent phenotypes of S. mutans YidC paralogs. Cardiolipin (CL), a prevalent phospholipid in the S. mutans cytoplasmic membrane during acid stress, is encoded by a single gene, cls. We show a concerted mechanism for cardiolipin and YidC2 under acid stress based on similarly increased promoter activities and similar elimination phenotypes. Using coarse grain molecular dynamics simulations with the Martini2.2 Forcefield, YidC1 and YidC2 wild-type and mutant interactions with CL were assessed in silico. We observed substantially increased CL interaction in dimeric versus monomeric proteins, and variable CL occupancy in YidC1 and YidC2 mutant constructs that mimicked characteristics of the other wild-type paralog. Hence, paralog-specific amino acid- CL interactions contribute to YidC1 and YidC2-associated phenotypes that can be exchanged by point mutation at positions 138 or 152, respectively.</p
DataSheet2_Cardiolipin occupancy profiles of YidC paralogs reveal the significance of respective TM2 helix residues in determining paralog-specific phenotypes.PDF
YidC belongs to an evolutionarily conserved family of insertases, YidC/Oxa1/Alb3, in bacteria, mitochondria, and chloroplasts, respectively. Unlike Gram-negative bacteria, Gram-positives including Streptococcus mutans harbor two paralogs of YidC. The mechanism for paralog-specific phenotypes of bacterial YidC1 versus YidC2 has been partially attributed to the differences in their cytoplasmic domains. However, we previously identified a W138R gain-of-function mutation in the YidC1 transmembrane helix 2. YidC1W138R mostly phenocopied YidC2, yet the mechanism remained unknown. Primary sequence comparison of streptococcal YidCs led us to identify and mutate the YidC1W138 analog, YidC2S152 to W/A, which resulted in a loss of YidC2- and acquisition of YidC1-like phenotype. The predicted lipid-facing side chains of YidC1W138/YidC2S152 led us to propose a role for membrane phospholipids in specific-residue dependent phenotypes of S. mutans YidC paralogs. Cardiolipin (CL), a prevalent phospholipid in the S. mutans cytoplasmic membrane during acid stress, is encoded by a single gene, cls. We show a concerted mechanism for cardiolipin and YidC2 under acid stress based on similarly increased promoter activities and similar elimination phenotypes. Using coarse grain molecular dynamics simulations with the Martini2.2 Forcefield, YidC1 and YidC2 wild-type and mutant interactions with CL were assessed in silico. We observed substantially increased CL interaction in dimeric versus monomeric proteins, and variable CL occupancy in YidC1 and YidC2 mutant constructs that mimicked characteristics of the other wild-type paralog. Hence, paralog-specific amino acid- CL interactions contribute to YidC1 and YidC2-associated phenotypes that can be exchanged by point mutation at positions 138 or 152, respectively.</p
Cooperative Gating of a K<sup>+</sup> Channel by Unmodified Biological Anionic Lipids Viewed by Solid-State NMR Spectroscopy
Lipids adhere to
membrane proteins to stimulate or suppress molecular
and ionic transport and signal transduction. Yet, the molecular details
of lipidāprotein interaction and their functional impact are
poorly characterized. Here we combine NMR, coarse-grained molecular
dynamics (CGMD), and functional assays to reveal classic cooperativity
in the binding and subsequent activation of a bacterial inward rectifier
potassium (Kir) channel by phosphatidylglycerol (PG), a common component
of many membranes. Past studies of lipid activation of Kir channels
focused primarily on phosphatidylinositol bisphosphate, a relatively
rare signaling lipid that is tightly regulated in space and time.
We use solid-state NMR to quantify the binding of unmodified 13C-PG to the K+ channel KirBac1.1 in liposomes.
This specific lipidāprotein interaction has a dissociation
constant (Kd) of ā¼7 mol percentage
PG (Ī§PG) with positive cooperativity (n = 3.8) and approaches saturation near 20% Ī§PG.
Liposomal flux assays show that K+ flux also increases
with PG in a cooperative manner with an EC50 of ā¼20%
Ī§PG, within the physiological range. Further quantitative
fitting of these data reveals that PG acts as a partial (80%) agonist
with fivefold K+ flux amplification. Comparisons of NMR
chemical shift perturbation and CGMD simulations at different Ī§PG confirm the direct interaction of PG with key residues,
several of which would not be accessible to lipid headgroups in the
closed state of the channel. Allosteric regulation by a common lipid
is directly relevant to the activation mechanisms of several human
ion channels. This study highlights the role of concentration-dependent
lipidāprotein interactions and tightly controlled protein allostery
in the activation and regulation of ion channels