Structure of the SLC4 transporter Bor1p in an inward‐facing conformation

Abstract

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at~6 Å resolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the Xray structure of AE1 in an outward-facing conformation, together with MD simulations of inwardfacing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily.

Evolution of substrate specificity in the Nucleobase-Ascorbate Transporter (NAT) protein family

“…The UraA and UapA monomers are made of mostly α-helical segments (TMS) characterized by a 7+7-helix inverted repeat 30 . Interestingly, the same topology is also found in transporters with very little primary amino acid sequence similarity with NATs, such as the AzgA-like purine transporters 36 the plant boron transporter Bor1 37 , the human Band3 anion exchanger 38 , or members of SulP transporter family 39 . All these are homodimeric transporters which seem to function via the so-called "elevator mechanism" of transport 40 .…”

Insights into the Mechanisms Underlying Boron Homeostasis in Plants

“…In addition, plants use BOR borate exporters for B exclusion from tissues under excess B conditions ( Miwa et al, 2007 ; Sutton et al, 2007 ; Schnurbusch et al, 2010 ). The BOR family has a similar structure to anion transporters ( Thurtle-Schmidt and Stroud, 2016 ; Coudray et al, 2017 ). A human BOR-like transporter, BTR1/SLC4A11, was characterized as an electrogenic, voltage-regulated Na + -coupled B(OH) 4 - cotransporter by electrophysiology in human embryonic kidney (HEK) 293 cells ( Park et al, 2004 ).…”

Asymmetry of inverted-topology repeats in the AE1 anion exchanger suggests an elevator-like mechanism

“…To gain insights into this mechanism, several studies have compared OF AE1 with structurally related transporters. Specifically, two recent studies have described low-resolution structures of SLC4-type boron transporters, namely a 4.1-Å-resolution crystal structure of occluded-state Bor1 from Arabidopsis thaliana ( Thurtle-Schmidt and Stroud, 2016 ), and a 6-Å-resolution structure of IF Saccharomyces mikatae Bor1p, obtained with cryoelectron microscopy ( Coudray et al, 2017 ). Confusingly, comparison of these structures with OF AE1 led to divergent conclusions, namely elevator-like in the former study ( Thurtle-Schmidt and Stroud, 2016 ) and rocking bundle in the latter ( Coudray et al, 2017 ).…”

“…Specifically, two recent studies have described low-resolution structures of SLC4-type boron transporters, namely a 4.1-Å-resolution crystal structure of occluded-state Bor1 from Arabidopsis thaliana ( Thurtle-Schmidt and Stroud, 2016 ), and a 6-Å-resolution structure of IF Saccharomyces mikatae Bor1p, obtained with cryoelectron microscopy ( Coudray et al, 2017 ). Confusingly, comparison of these structures with OF AE1 led to divergent conclusions, namely elevator-like in the former study ( Thurtle-Schmidt and Stroud, 2016 ) and rocking bundle in the latter ( Coudray et al, 2017 ). Divergent conclusions have also resulted from comparisons of the AE1 structure with the IF structures of SLC26Dg ( Chang and Geertsma, 2017 ; elevator-like) and the SLC23-family UraA transporter ( Reithmeier et al, 2016 ; rocking bundle).…”