Structural Features Mediating Zinc Binding and Transfer in the AztABCD Zinc Transporter System.

Anusha Meni,Erik T Yukl

doi:10.3390/biom10081156

Abstract

Many bacteria require ATP binding cassette (ABC) transporters for the import of the essential metal zinc from limited environments. These systems rely on a periplasmic or cell-surface solute binding protein (SBP) to bind zinc with high affinity and specificity. AztABCD is one such zinc transport system recently identified in a large group of diverse bacterial species. In addition to a classical SBP (AztC), the operon also includes a periplasmic metallochaperone (AztD) shown to transfer zinc directly to AztC. Crystal structures of both proteins from Paracoccus denitrificans have been solved and suggest several structural features on each that may be important for zinc binding and transfer. Here we determine zinc binding affinity, dissociation kinetics, and transfer kinetics for several deletion mutants as well as a crystal structure for one of them. The results indicate specific roles for loop structures on AztC and an N-terminal motif on AztD in zinc binding and transfer. These data are consistent with a structural transfer model proposed previously and provide further mechanistic insight into the processes of zinc binding and transfer.

Highlights

Transition metals such as zinc, iron, manganese, and copper provide essential cellular functions, yet are highly toxic in their “free” forms or bound to incorrect targets
The zinc binding affinity of WT AztD, WT AztC, ∆D-Loop AztC, and ∆Z-Loop AztC were previously determined by competition assay with the fluorophore MagFura-2 (MF2) (Table 1)
We apply this technique to H138/204A AztC and a deletion of the N-terminal motif in AztD (∆N-terminal Motif (NTM) AztD) (Figure 2)

Summary

Introduction

Transition metals such as zinc, iron, manganese, and copper provide essential cellular functions, yet are highly toxic in their “free” forms or bound to incorrect targets. All living things must carefully maintain metal homeostasis This is accomplished by regulating the uptake and efflux of metals from the cell as well as by proteins that bind, store, and/or shuttle metals to their appropriate destinations. Proteins of the latter description are known as metallochaperones. A new family of intracellular zinc-binding GTPases called COG0523 has been identified in bacteria. These are often upregulated by zinc starvation [4,5,6] and may function as metallochaperones. Direct evidence of metal transfer to target proteins has never been demonstrated, and their physiological functions are often unclear

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Conclusion