Multi-genomic analysis of the Cation Diffusion Facilitator transporters from algae

AbstractMetal transport processes are relatively poorly understood in algae in comparison to higher plants and other eukaryotes. A screen of genomes from 33 taxonomically diverse algal species was conducted to identify members of the Cation Diffusion Facilitator (CDF) family of metal ion transporter. All algal genomes contained at least one CDF gene with four species having >10 CDF genes (median of 5 genes per genome), further confirming that this is a ubiquitous gene family. Phylogenetic analysis suggested a CDF gene organisation of five groups, which includes Zn-CDF, Fe/Zn-CDF and Mn-CDF groups, consistent with previous phylogenetic analyses, and two functionally undefined groups. One of these undefined groups was algal specific although excluded chlorophyte and rhodophyte sequences. The majority of sequences (22 out of 26 sequences) from this group had a putative ion binding site motif within transmembrane domain 2 and 5 that was distinct from other CDF proteins, such that alanine or serine replaced the conserved histidine residue. The phylogenetic grouping was supported by sequence cluster analysis. Yeast heterologous expression of CDF proteins from Chlamydomonas reinhardtii indicated Zn2+ and Co2+ transport function by CrMTP1, and Mn2+ transport function by CrMTP2, CrMTP3 and CrMTP4, which validated the phylogenetic prediction. However, the Mn-CDF protein CrMTP3 was also able to provide zinc and cobalt tolerance to the Zn- and Co-sensitive zrc1cot1 yeast strain. There is wide diversity of CDF transporters within the algae lineage, and some of these genes may be attractive targets for future applications of metal content engineering in plants or microorganisms.

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Functional Determinants of Metal Ion Transport and Selectivity in Paralogous Cation Diffusion Facilitator Transporters CzcD and MntE in Streptococcus pneumoniae

Journal of Bacteriology ◽

10.1128/jb.00975-15 ◽

2016 ◽

Vol 198 (7) ◽

pp. 1066-1076 ◽

Cited By ~ 25

Author(s):

Julia E. Martin ◽

David P. Giedroc

Keyword(s):

Streptococcus Pneumoniae ◽

Metal Ion ◽

Transmembrane Domain ◽

Divalent Metal ◽

Cation Diffusion ◽

Metal Specificity ◽

Cation Diffusion Facilitator ◽

Content Type ◽

Metal Selectivity ◽

A Site

ABSTRACTCation diffusion facilitators (CDFs) are a large family of divalent metal transporters that collectively possess broad metal specificity and contribute to intracellular metal homeostasis and virulence in bacterial pathogens.Streptococcus pneumoniaeexpresses two homologous CDF efflux transporters, MntE and CzcD. Cells lackingmntEorczcDare sensitive to manganese (Mn) or zinc (Zn) toxicity, respectively, and specifically accumulate Mn or Zn, respectively, thus suggesting that MntE selectively transports Mn, while CzcD transports Zn. Here, we probe the origin of this metal specificity using a phenotypic growth analysis of pneumococcal variants. Structural homology toEscherichia coliYiiP predicts that both MntE and CzcD are dimeric and each protomer harbors four pairs of conserved metal-binding sites, termed the A site, the B site, and the C1/C2 binuclear site. We find that single amino acid mutations within both the transmembrane domain A site and the B site in both CDFs result in a cellular metal sensitivity similar to that of the corresponding null mutants. However, multiple mutations in the predicted cytoplasmic C1/C2 cluster of MntE have no impact on cellular Mn resistance, in contrast to the analogous substitutions in CzcD, which do have on impact on cellular Zn resistance. Deletion of the MntE-specific C-terminal tail, present only in Mn-specific bacterial CDFs, resulted in only a modest growth phenotype. Further analysis of MntE-CzcD functional chimeric transporters showed that Asn and Asp in theND-DD A-site motif of MntE and the most N-terminal His in theHD-HD site A of CzcD (the specified amino acids are underlined) play key roles in transporter metal selectivity.IMPORTANCECation diffusion facilitator (CDF) proteins are divalent metal ion transporters that are conserved in organisms ranging from bacteria to humans and that play important roles in cellular physiology, from metal homeostasis and resistance to type I diabetes in vertebrates. The respiratory pathogenStreptococcus pneumoniaeexpresses two metal CDF transporters, CzcD and MntE. How CDFs achieve metal selectivity is unclear. We show here that CzcD and MntE are true paralogs, as CzcD transports zinc, while MntE selectively transports manganese. Through the use of an extensive collection of pneumococcal variants, we show that a primary determinant for metal selectivity is the A site within the transmembrane domain. This extends our understanding of how CDFs discriminate among transition metals.

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Genome-Wide Identification and Expression Analysis of the Cation Diffusion Facilitator Gene Family in Turnip Under Diverse Metal Ion Stresses

Frontiers in Genetics ◽

10.3389/fgene.2018.00103 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 10

Author(s):

Xiong Li ◽

Yuansheng Wu ◽

Boqun Li ◽

Wenqi He ◽

Yonghong Yang ◽

...

Keyword(s):

Gene Family ◽

Expression Analysis ◽

Metal Ion ◽

Cation Diffusion ◽

Cation Diffusion Facilitator ◽

Genome Wide

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A Preliminary Bioinformatics Analysis of Cation Diffusion Facilitator (CDF) Proteins in Different Plants

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.282-283.453 ◽

2011 ◽

Vol 282-283 ◽

pp. 453-456

Author(s):

Hua Zhong ◽

Bao Ping Sun ◽

Fang Ying Zhao

Keyword(s):

Secretory Pathway ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Populus Trichocarpa ◽

Alpha Helix ◽

Random Coil ◽

Post Translational Modification ◽

Cation Diffusion ◽

Cation Diffusion Facilitator ◽

Arabidopsis Lyrata

The amino acid sequence of Cation Diffusion Facilitator from Populus trichocarpa, Thlaspi goesingense, Arabidopsis lyrata subsp. Lyrata, Brassica juncea and Medicago sativa, registered in GenBank, were analyzed and researched by the bioinformatic tools in the several aspects, including hydrophobicity / hydrophilicity properties, post-translational modification, secondary structures prediction and transmembrane domain. The results showed that Cation Diffusion Facilitator is a hydrophobic and transmembrane protein, which exists in endoplasmic reticulum and other secretory pathway. The main motifs of predicted secondary structure of Cation Diffusion Facilitator are alpha helix and random coil.

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The cation diffusion facilitator protein MamM's cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014145 ◽

2020 ◽

Vol 295 (49) ◽

pp. 16614-16629

Author(s):

Shiran Barber-Zucker ◽

Jenny Hall ◽

Afonso Froes ◽

Sofiya Kolusheva ◽

Fraser MacMillan ◽

...

Keyword(s):

Transmembrane Domain ◽

Magnetotactic Bacteria ◽

Crystal Form ◽

Binding Modes ◽

Cation Diffusion ◽

Cation Diffusion Facilitator ◽

Metal Type ◽

Metal Selectivity ◽

Functional Discrimination ◽

Metal Recognition

Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain, in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal or multiple metals from the cytoplasm, and it is not known whether the CTD takes an active regulatory role in metal recognition and discrimination during cation transport. Here, the model CDF protein MamM, an iron transporter from magnetotactic bacteria, was used to probe the role of the CTD in metal recognition and selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of their binding sites, thermodynamics, and binding-dependent conformations, both in crystal form and in solution, which suggests a varying level of functional discrimination between CDF domains. Furthermore, these results provide the first direct evidence that CDF CTDs play a role in metal selectivity. We demonstrate that MamM's CTD can discriminate against Mn2+, supporting its postulated role in preventing magnetite formation poisoning in magnetotactic bacteria via Mn2+ incorporation.

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The cation diffusion facilitator protein MamM’s cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+

10.1101/2020.02.02.930644 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shiran Barber-Zucker ◽

Jenny Hall ◽

Afonso Froes ◽

Sofiya Kolusheva ◽

Fraser MacMillan ◽

...

Keyword(s):

Direct Evidence ◽

Transmembrane Domain ◽

Crystal Form ◽

Binding Modes ◽

Cation Diffusion ◽

Cation Diffusion Facilitator ◽

Metal Type ◽

Metal Selectivity ◽

Specific Metal

SummaryCation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain (TMD), in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal, or multiple metals, from the cytoplasm. Here, the model CDF protein MamM, from magnetotactic bacteria, was used to probe the role of the CTD in metal selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of; their binding sites, thermodynamics and binding-dependent conformation, both in crystal form and in solution. Furthermore, the results indicate that the CTD discriminates against Mn2+ and provides the first direct evidence that CDF CTD’s play a role in metal selectivity.

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Putative metal binding site in the transmembrane domain of the manganese transporter SLC30A10 is different from that of related zinc transporters

Metallomics ◽

10.1039/c8mt00115d ◽

2018 ◽

Vol 10 (8) ◽

pp. 1053-1064 ◽

Cited By ~ 7

Author(s):

Charles E. Zogzas ◽

Somshuvra Mukhopadhyay

Keyword(s):

Binding Site ◽

Metal Binding ◽

Transmembrane Domain ◽

Zinc Transporters ◽

Cation Diffusion ◽

Metal Binding Site ◽

Cation Diffusion Facilitator

Mechanism by which the cation diffusion facilitator SLC30A10 transports manganese is fundamentally different from that of previously-studied proteins in this superfamily.

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The metal binding site composition of the cation diffusion facilitator protein MamM cytoplasmic domain impacts its metal responsivity

10.1101/2020.02.06.936617 ◽

2020 ◽

Author(s):

Shiran Barber-Zucker ◽

Anat Shahar ◽

Sofiya Kolusheva ◽

Raz Zarivach

Keyword(s):

Metal Binding ◽

Binding Sites ◽

Transmembrane Domain ◽

Metal Cations ◽

Data Bank ◽

Cation Diffusion ◽

Cation Diffusion Facilitator ◽

Metal Selectivity ◽

The Impact

AbstractThe cation diffusion facilitator (CDF) is a conserved family of divalent d-block metal cation transporters that extrude these cations selectively from the cytoplasm. CDF proteins are composed of two domains: the transmembrane domain, through which the cations are transported, and a regulatory cytoplasmic C-terminal domain (CTD). Metal binding to the CTD leads to its tighter conformation, and this sequentially promotes conformational change of the transmembrane domain which allows the actual transport of specific metal cations. It was recently shown that the magnetotactic bacterial CDF protein MamM CTD has a role in metal selectivity, as binding of different metal cations exhibits distinctive affinities and conformations. It is yet unclear whether the composition of the CTD binding sites can impact metal selectivity. Here we performed a mutational study of MamM CTD, where we exchanged the metal binding residues with different metal-binding amino acids. Using X-ray crystallography and Trp-fluorescence spectrometry, we studied the impact of the mutations on the CTD conformation in the presence of different metals. Our results reveal that the incorporation of such mutations alters the domain response to metals in vitro, as mutant forms of the CTD bind metals differently in terms of the composition of the binding sites and the CTD conformation.CoordinatesMamM CTD structures have been deposited in the Protein Data Bank under the following accession codes: 6H5V, 6H5M, 6H5U, 6H8G, 6HAO, 6H88, 6H87, 6H8A, 6H89, 6H8D, 6H5K, 6H9Q, 6H84, 6H83, 6HA2, 6H8I, 6H9T, 6H81, 6HAN, 6H85, 6H9P, 6HHS.

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Multi-genomic analysis of the cation diffusion facilitator transporters from algae

Metallomics ◽

10.1039/d0mt00009d ◽

2020 ◽

Vol 12 (4) ◽

pp. 617-630 ◽

Cited By ~ 2

Author(s):

Aniefon Ibuot ◽

Andrew P. Dean ◽

Jon K. Pittman

Keyword(s):

Functional Analysis ◽

Substrate Specificity ◽

Chlamydomonas Reinhardtii ◽

Genomic Analysis ◽

Cation Diffusion ◽

Metal Transporters ◽

Cation Diffusion Facilitator

Cation diffusion facilitator metal transporters are widespread throughout algae and include a novel algal-specific clade. Functional analysis of Chlamydomonas reinhardtii isoforms partly validated phylogenetic prediction of substrate specificity.

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Structural basis for the alternating access mechanism of the cation diffusion facilitator YiiP

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715051115 ◽

2018 ◽

Vol 115 (12) ◽

pp. 3042-3047 ◽

Cited By ~ 19

Author(s):

Maria Luisa Lopez-Redondo ◽

Nicolas Coudray ◽

Zhening Zhang ◽

John Alexopoulos ◽

David L. Stokes

Keyword(s):

Large Scale ◽

Structural Basis ◽

Membrane Domains ◽

Cation Diffusion ◽

Cation Diffusion Facilitator ◽

X Ray ◽

Bacterial Membranes ◽

Helix Bundle ◽

Four Helix Bundle ◽

Alternating Access

YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn2+ across bacterial membranes. Previous work defined the atomic structure of an outward-facing conformation, the location of several Zn2+ binding sites, and hydrophobic residues that appear to control access to the transport sites from the cytoplasm. A low-resolution cryo-EM structure revealed changes within the membrane domain that were associated with the alternating access mechanism for transport. In the current work, the resolution of this cryo-EM structure has been extended to 4.1 Å. Comparison with the X-ray structure defines the differences between inward-facing and outward-facing conformations at an atomic level. These differences include rocking and twisting of a four-helix bundle that harbors the Zn2+ transport site and controls its accessibility within each monomer. As previously noted, membrane domains are closely associated in the dimeric structure from cryo-EM but dramatically splayed apart in the X-ray structure. Cysteine crosslinking was used to constrain these membrane domains and to show that this large-scale splaying was not necessary for transport activity. Furthermore, dimer stability was not compromised by mutagenesis of elements in the cytoplasmic domain, suggesting that the extensive interface between membrane domains is a strong determinant of dimerization. As with other secondary transporters, this interface could provide a stable scaffold for movements of the four-helix bundle that confers alternating access of these ions to opposite sides of the membrane.

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