Dataset Information

Fine-tuning spermidine binding modes in the putrescine binding protein PotF.

ABSTRACT: A profound understanding of the molecular interactions between receptors and ligands is important throughout diverse research, such as protein design, drug discovery, or neuroscience. What determines specificity and how do proteins discriminate against similar ligands? In this study, we analyzed factors that determine binding in two homologs belonging to the well-known superfamily of periplasmic binding proteins, PotF and PotD. Building on a previously designed construct, modes of polyamine binding were swapped. This change of specificity was approached by analyzing local differences in the binding pocket as well as overall conformational changes in the protein. Throughout the study, protein variants were generated and characterized structurally and thermodynamically, leading to a specificity swap and improvement in affinity. This dataset not only enriches our knowledge applicable to rational protein design but also our results can further lay groundwork for engineering of specific biosensors as well as help to explain the adaptability of pathogenic bacteria.

SUBMITTER: Kroger P

PROVIDER: S-EPMC8666671 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Fine-tuning spermidine binding modes in the putrescine binding protein PotF.

Kröger Pascal P Shanmugaratnam Sooruban S Scheib Ulrike U Höcker Birte B

The Journal of biological chemistry 20211119 6

A profound understanding of the molecular interactions between receptors and ligands is important throughout diverse research, such as protein design, drug discovery, or neuroscience. What determines specificity and how do proteins discriminate against similar ligands? In this study, we analyzed factors that determine binding in two homologs belonging to the well-known superfamily of periplasmic binding proteins, PotF and PotD. Building on a previously designed construct, modes of polyamine bind ...[more]

PMID: 34801550

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Project description:The mechanism of mammalian polyamine transport is poorly understood. We have investigated the role of plasma-membrane potential (DeltaPsipm) in putrescine and spermidine uptake in ZR-75-1 human breast cancer cells. The rate of [3H]putrescine and [3H]spermidine uptake was inversely correlated to extracellular [K+] ([K+]o) and to DeltaPsipm, as determined by the accumulation of [3H]tetraphenylphosphonium bromide (TPP). Inward transport was unaffected by a selective decrease in mitochondrial potential (DeltaPsimit) induced by valinomycin at low [K+]o, but was reduced by approximately 60% by the rheogenic protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which rapidly (<=15 min) collapsed both DeltaPsipm and DeltaPsimit. Plasma-membrane depolarization by high [K+]o or CCCP did not enhance putrescine efflux in cells pre-loaded with [3H]putrescine, suggesting that decreased uptake caused by these agents did not result from a higher excretion rate. On the other hand, the electroneutral K+/H+ exchanger nigericin (10 microM) co-operatively depressed -3H-TPP, [3H]putrescine and [3H]spermidine uptake in the presence of ouabain. Suppression of putrescine uptake by nigericin+ouabain was Na+-dependent, suggesting that plasma-membrane repolarization by the electrogenic Na+ pump was required upon acidification induced by nigericin, due to the activation of the Na+/H+ antiporter. The sole addition of 5-N, N-hexamethylene amiloride, a potent inhibitor of the Na+/H+ antiporter, strongly inhibited putrescine uptake in a competitive fashion -Ki 4.0+/-0.9 (S.D.) microM-, while being a weaker antagonist of spermidine uptake. The potency of a series of amiloride analogues to inhibit putrescine uptake was clearly different from that of the Na+/H+ antiporter, and resembled that noted for Na+ co-transport proteins. These data demonstrate that putrescine and spermidine influx is mainly unidirectional and strictly depends on DeltaPsipm, but not DeltaPsimit. This report also provides first evidence for a high-affinity amiloride-binding site on the putrescine carrier, which provides new insight into the biochemical properties of this transporter.

Dataset Information

Fine-tuning spermidine binding modes in the putrescine binding protein PotF.

Publications

Fine-tuning spermidine binding modes in the putrescine binding protein PotF.

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