Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters

Abstract

Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.

Distinct specific interactions of the UapA transporter with membrane lipids are critical for dimerization, ER-exit and function

“…Apparently, mutation K73A/R133A/R421A was more critical than R287A/R478A/R479A for the packaging of UapA into COPII secretory vesicles and ER-exit [6], which might in turn suggest that annular lipid interactions are more important for the trafficking of UapA and other structurally similar eukaryotic transporters. In line with our results, it has been recently suggested that lipid binding around domain interfaces of the prokaryotic NhaA Na+/H + exchanger are also involved in stabilizing the core domain during the conformational transitions required for transport by the elevator mechanism [38]. It has thus been speculated that elevator-type antiporters use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.…”

“…It is becoming well-established that the physicochemical nature of lipid bilayers and specific lipid composition of membranes affect transporter folding, oligomerization, subcellular trafficking, function and turnover [8,[32][33][34][35][36][37][38]. For transporters conforming to the 5+5 inverted repeat (IR) or LeuT fold, similarities in structurally resolved lipidprotein interactions suggest common ways in which transporter structure and function are supported by lipid interactions [39].…”

Magnifying ion mobility spectrometry–mass spectrometry measurements for biomolecular structure studies

“…al . The researchers employed the IMPACT software to aid in the modeling of the Na + /H + exchanger NapA from Thermus thermophiles [76]. Results from the gas-phase study suggest that lipid interactions stabilize the native structure.…”

Mass spectrometry-enabled structural biology of membrane proteins

“…This is related to the strength of the oligomerization interface, with complexes comprising interfaces with low buried surface areas and few salt bridges requiring lipid binding for stabilization [22]. Additionally, the binding of lipids that have been carried through purification protocols has been observed in native mass spectra for several MPs [20,21,89,129]. For example, in the case of the F 0 F 1 ATPase from Enterococcus hirae, the membrane embedded ring was identified as a decamer with ten cardiolipin molecules bound.…”

“…Combining IMS with low resolution modelling [145], conventional, or steered molecular dynamics (MD) simulations [81,129] is also gaining traction as a method for obtaining structural insights into proteins/assemblies. IMS also enables structural perturbations, for example, as a result of ligand binding, to be studied (see below).…”