Selective Translocation of Cyclic Sugars through Dynamic Bacterial Transporter

Abstract

The selective translocation of molecules through membrane pores is an integral process in cells. We present a bacterial sugar transporter, CymA of unusual structural conformation due to a dynamic N terminus segment in the pore, reducing its diameter. We quantified the translocation kinetics of various cyclic sugars of different charge, size, and symmetry across native and truncated CymA devoid of the N terminus using singlechannel recordings. The chemically divergent cyclic hexasaccharides bind to the native and truncated pore with high affinity and translocate effectively. Specifically, these sugars bind and translocate rapidly through truncated CymA compared to native CymA. In contrast, larger cyclic heptasaccharides and octasaccharides do not translocate but bind to native and truncated CymA with distinct binding kinetics highlighting the importance of molecular charge, size and symmetry in translocation consistent with liposome assays. Based on the sugar-binding kinetics, we suggest that the N terminus most likely resides inside the native CymA barrel, regulating the transport rate of cyclic sugars. Finally, we present native CymA as a large nanopore sensor for the simultaneous single-molecule detection of various sugars at high resolution, establishing its functional versatility. This natural pore is expected to have several applications in nanobiotechnology and will help further our understanding of the fundamental mechanism of molecular transport.

Electrostatic Filtering of Polypeptides Through Membrane Protein Pores

“…This data at low salt buffer indicates weak electrostatic interaction of D9 peptides with few positively charged residues at the trans side of the pore as opposed to no D9 binding at high salt buffer, confirming the salt‐dependent charge screening effect. The addition of R9 peptides to the same pore produced well‐defined ion current blockages and this competitive binding of D9 and R9 peptides confirmed the charge‐selective translocation of cationic peptides through the CymA pores in agreement with the cation‐selective nature of the pore (Figure 4c) [47,49] …”

“…Previous studies have shown that CymA is naturally evolved for the selective uptake of six‐fold symmetric cyclic hexasaccharide, which binds to specific charged residues in the pore with high binding affinity [47,49] . This pore has a unique N terminal segment that has not been resolved by X‐ray crystallography due to its dynamic nature and its role in sensing is uncertain [46] .…”

“…CymA also showed clear asymmetry in charge distribution along the pore lining, containing numerous negatively charged and few positively charged residues [46] . Previously we have shown that these densely packed charged residues at the pore lumen act as an affinity site facilitating the binding and translocation of both neutral and charged cyclic hexasaccharides [47,49] . Specifically, charged CDs are electrophoretically driven through the pore, whereas neutral CDs undergo electroosmotic‐driven translocation [47,48] .…”

“…Here, we introduce a natural pore CymA of an unusual architecture derived from the outer membrane of Klebsiella oxytoca , which is specific for the passive uptake of bulky cyclic sugars [45–49] . Several membrane porins possess extracellular loops; however, in CymA, the N‐terminus periplasmic segment constricts the pore diameter and generates a unique pore conformation [38,46] .…”

“…[10,23] Here, we introduce a natural pore CymA of an unusual architecture derived from the outer membrane of Klebsiella oxytoca, which is specific for the passive uptake of bulky cyclic sugars. [45][46][47][48][49] Several membrane porins possess extracellular loops; however, in CymA, the N-terminus periplasmic segment constricts the pore diameter and generates a unique pore conformation. [38,46] Remarkably, the structure of the co-crystal of CymA and alpha-cyclodextrin (CD) revealed two binding sites, one at the pore entrance and another at the exit site, revealing a unique molecular transport pathway.…”