Designed High-Redox Potential Laccases Exhibit High Functional Diversity.

White-rot fungi secrete an impressive repertoire of high-redox potential laccases (HRPLs) and peroxidases for efficient oxidation and utilization of lignin. Laccases are attractive enzymes for the chemical industry due to their broad substrate range and low environmental impact. Since expression of functional recombinant HRPLs is challenging, however, iterative-directed evolution protocols have been applied to improve their expression, activity, and stability. We implement a rational, stabilize-and-diversify strategy to two HRPLs that we could not functionally express. First, we use the PROSS stability-design algorithm to allow functional expression in yeast. Second, we use the stabilized enzymes as starting points for FuncLib active-site design to improve their activity and substrate diversity. Four of the FuncLib-designed HRPLs and their PROSS progenitor exhibit substantial diversity in reactivity profiles against high-redox potential substrates, including lignin monomers. Combinations of 3-4 subtle mutations that change the polarity, solvation, and sterics of the substrate-oxidation site result in orders of magnitude changes in reactivity profiles. These stable and versatile HRPLs are a step toward generating an effective lignin-degrading consortium of enzymes that can be secreted from yeast. The stabilize-and-diversify strategy can be applied to other challenging enzyme families to study and expand the utility of natural enzymes.

Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences.

White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability, and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning-based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wildtype enzymes are functionally expressed in yeast, whereas their wildtype parents are not. Three of these designs exhibit substantial and useful diversity in their reactivity profiles and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families directly from genomic databases.

Loss-of-function SLC30A2 mutants are associated with gut dysbiosis and alterations in intestinal gene expression in preterm infants.

Loss of Paneth cell (PC) function is implicated in intestinal dysbiosis, mucosal inflammation, and numerous intestinal disorders, including necrotizing enterocolitis (NEC). Studies in mouse models show that zinc transporter ZnT2 (SLC30A2) is critical for PC function, playing a role in granule formation, secretion, and antimicrobial activity; however, no studies have investigated whether loss of ZnT2 function is associated with dysbiosis, mucosal inflammation, or intestinal dysfunction in humans. SLC30A2 was sequenced in healthy preterm infants (26-37 wks; n = 75), and structural analysis and functional assays determined the impact of mutations. In human stool samples, 16S rRNA sequencing and RNAseq of bacterial and human transcripts were performed. Three ZnT2 variants were common (>5%) in this population: H346Q, f = 19%; L293R, f = 7%; and a previously identified compound substitution in Exon7, f = 16%). H346Q had no effect on ZnT2 function or beta-diversity. Exon7 impaired zinc transport and was associated with a fractured gut microbiome. Analysis of microbial pathways suggested diverse effects on nutrient metabolism, glycan biosynthesis and metabolism, and drug resistance, which were associated with increased expression of host genes involved in tissue remodeling. L293R caused profound ZnT2 dysfunction and was associated with overt gut dysbiosis. Microbial pathway analysis suggested effects on nucleotide, amino acid and vitamin metabolism, which were associated with the increased expression of host genes involved in inflammation and immune response. In addition, L293R was associated with reduced weight gain in the early postnatal period. This implicates ZnT2 as a novel modulator of mucosal homeostasis in humans and suggests that genetic variants in ZnT2 may affect the risk of mucosal inflammation and intestinal disease.

Metal transport mechanism of the cation diffusion facilitator (CDF) protein family - a structural perspective on human CDF (ZnT)-related diseases.

Divalent d-block metal cations (DDMCs) participate in many cellular functions; however, their accumulation in cells can be cytotoxic. The cation diffusion facilitator (CDF) family is a ubiquitous family of transmembrane DDMC exporters that ensures their homeostasis. Severe diseases, such as type II diabetes, Parkinson's and Alzheimer's disease, were linked to dysfunctional human CDF proteins, ZnT-1-10 (SLC30A1-10). Each member of the CDF family reduces the cytosolic concentration of a specific DDMC by transporting it from the cytoplasm to the extracellular environment or into intracellular compartments. This process is usually achieved by utilizing the proton motive force. In addition to their activity as DDMC transporters, CDFs also have other cellular functions such as the regulation of ion channels and enzymatic activity. The combination of structural and biophysical studies of different bacterial and eukaryotic CDF proteins led to significant progress in the understanding of the mutual interaction among CDFs and DDMCs, their involvement in ion binding and selectivity, conformational changes and the consequent transporting mechanisms. Here, we review these studies, provide our mechanistic interpretation of CDF proteins based on the current literature and relate the above to known human CDF-related diseases. Our analysis provides a common structure-function relationship to this important protein family and closes the gap between eukaryote and prokaryote CDFs.

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

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.

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

The 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). It was recently shown that the CTD of the CDF protein MamM from magnetotactic bacteria 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 and if we can manipulate the CTD to response to other non-native metals in CDF proteins. Here we performed a mutational study of the model protein MamM CTD, where we exchanged the native metal binding residues with different metal-binding amino acids. Using X-ray crystallography and Trp-fluorescence spectrometry, we studied the impact of these mutations on the CTD conformation in the presence of non-native 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. Therefore, the results demonstrate the direct influence of the CTD binding site composition on CDF proteins structure and hence, function, and constitute a first step for rational design of MamM for transporting different metals in vivo.

A common genetic variant in zinc transporter ZnT2 (Thr288Ser) is present in women with low milk volume and alters lysosome function and cell energetics.

Suboptimal lactation is a common, yet underappreciated cause for early cessation of breastfeeding. Molecular regulation of mammary gland function is critical to the process lactation; however, physiological factors underlying insufficient milk production are poorly understood. The zinc (Zn) transporter ZnT2 is critical for regulation of mammary gland development and maturation during puberty, lactation, and postlactation gland remodeling. Numerous genetic variants in the gene encoding ZnT2 (SLC30A2) are associated with low milk Zn concentration and result in severe Zn deficiency in exclusively breastfed infants. However, the functional impacts of genetic variation in ZnT2 on key mammary epithelial cell functions have not yet been systematically explored at the cellular level. Here we determined a common mutation in SLC30A2/ZnT2 substituting serine for threonine at amino acid 288 (Thr288Ser) was found in 20% of women producing low milk volume (n = 2/10) but was not identified in women producing normal volume. Exploration of cellular consequences in vitro using phosphomimetics showed the serine substitution promoted preferential phosphorylation of ZnT2, driving localization to the lysosome and increasing lysosome biogenesis and acidification. While the substitution did not initiate lysosome-mediated cell death, cellular ATP levels were significantly reduced. Our findings demonstrate the Thr288Ser mutation in SLC30A2/ZnT2 impairs critical functions of mammary epithelial cells and suggest a role for genetic variation in the regulation of milk production and lactation performance.

Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain.

BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Like other cytotoxicity-mediating effectors, BteA uses its multifunctional N-terminal domain to target phosphatidylinositol (PI)-rich microdomains in the host membrane. Despite their structural similarity, T3SS effectors exhibit a variable range of membrane interaction modes, and currently only limited structural information is available for the BteA membrane-targeting domain and the molecular mechanisms underlying its function. Employing a synergistic combination of structural methods, here we determine the structure of this functional domain and uncover key molecular determinants mediating its interaction with membranes. Residues 29-121 of BteA form an elongated four-helix bundle packed against two shorter perpendicular helices, the second of which caps the domain in a critical 'tip motif'. A flexible region preceding the BteA helical bundle contains the characteristic ß-motif required for binding its cognate chaperone BtcA. We show that BteA targets PI(4,5)P2-containing lipoprotein nanodiscs and binds a soluble PI(4,5)P2 analog via an extensive positively charged surface spanning its first two helices, and that this interaction is weaker for PI(3,5)P2 and abolished for PI(4)P. We confirmed this model of membrane-targeting by observation of BteA-induced changes in the structure of PI(4,5)P2-containing phospholipid bilayers using small-angle X-ray scattering (SAXS). We also extended these results to a larger BteA domain (residues 1-287), confirming its interaction with bilayers using calorimetry, fluorescence and SAXS methods. This novel view of the structural underpinnings of membrane targeting by BteA is an important step towards a comprehensive understanding of cytotoxicity in Bordetella, as well as interactions of a broad range of pathogens with their respective hosts.

Metal binding to the dynamic cytoplasmic domain of the cation diffusion facilitator (CDF) protein MamM induces a 'locked-in' configuration.

Cation diffusion facilitator (CDF) proteins are a conserved family of transmembrane transporters that ensure cellular homeostasis of divalent transition metal cations. Metal cations bind to CDF protein's cytoplasmic C-terminal domain (CTD), leading to closure from its apo open V-shaped dimer to a tighter packed structure, followed by a conformational change of the transmembrane domain, thus enabling transport of the metal cation. By implementing a comprehensive range of biochemical and biophysical methods, we studied the molecular mechanism of metal binding to the magnetotactic bacterial CDF protein MamM CTD. Our results reveal that the CTD is rather dynamic in its apo form, and that two dependent metal-binding sites, a single central binding site and two symmetrical, peripheral sites, are available for metal binding. However, only cation binding to the peripheral sites leads to conformational changes that lock the protein in a compact state. Thus, this work reveals how metal binding is regulating the sequential uptakes of metal cations by MamM, and extends our understanding of the complex regulation mechanism of CDF proteins. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession numbers: 6G64, 6G55, 6G5E and 6G6I (for CS, C267S, CS-C267S and W247A, respectively).

Zinc transporter 10 (ZnT10)-dependent extrusion of cellular Mn2+ is driven by an active Ca2+-coupled exchange.

Manganese (Mn2+) is extruded from the cell by the zinc transporter 10 (ZnT10). Loss of ZnT10 expression caused by autosomal mutations in the ZnT10 gene leads to hypermanganesemia in multiple organs. Here, combining fluorescent monitoring of cation influx in HEK293-T cells expressing human ZnT10 with molecular modeling of ZnT10 cation selectivity, we show that ZnT10 is exploiting the transmembrane Ca2+ inward gradient for active cellular exchange of Mn2+ In analyzing ZnT10 activity we used the ability of Fura-2 to spectrally distinguish between Mn2+ and Ca2+ fluxes. We found that (a) application of Mn2+-containing Ca2+-free solution to ZnT10-expressing cells triggers an influx of Mn2+, (b) reintroduction of Ca2+ leads to cellular Mn2+ extrusion against an inward Mn2+ gradient, and (c) the cellular transport of Mn2+ by ZnT10 is coupled to a reciprocal movement of Ca2+ Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn2+/Ca2+ exchange to an uncoupled channel mode, permeable to both Ca2+ and Mn2+ The findings in our study identify the first ion transporter that uses the Ca2+ gradient for active counter-ion exchange. They highlight a remarkable versatility in metal selectivity and mode of transport controlled by the tetrahedral metal transport site of ZnT proteins.

Transition metal binding selectivity in proteins and its correlation with the phylogenomic classification of the cation diffusion facilitator protein family.

Divalent d-block metal cations (DDMCs), such as Fe, Zn and Mn, participate in many biological processes. Understanding how specific DDMCs are transported to and within the cell and what controls their binding selectivity to different proteins is crucial for defining the mechanisms of metalloproteins. To better understand such processes, we scanned the RCSB Protein Data Bank, performed a de novo structural-based comprehensive analysis of seven DDMCs and found their amino acid binding and coordination geometry propensities. We then utilized these results to characterize the correlation between metal selectivity, specific binding site composition and phylogenetic classification of the cation diffusion facilitator (CDF) protein family, a family of DDMC transporters found throughout evolution and sharing a conserved structure, yet with different members displaying distinct metal selectivity. Our analysis shows that DDMCs differ, at times significantly, in terms of their binding propensities, and that in each CDF clade, the metal selectivity-related binding site has a unique and conserved sequence signature. However, only limited correlation exists between the composition of the DDMC binding site in each clade and the metal selectivity shown by its proteins.

SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome.

A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an ∼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4.4 at D4S2971; θ = 0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus, segregating as expected within the kindred and not found in a homozygous state in 300 Bedouin controls. We showed that SLC30A9 (solute carrier family 30 member 9; also known as ZnT-9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH-SY5Y cells overexpressing SLC30A9 fused to enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum, not co-localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signalling. However, using dual-luciferase reporter assay in SH-SY5Y cells we showed that Wnt signalling was not affected by the mutation. Based on protein modelling, the identified mutation is expected to affect SLC30A9's highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn2+ measurements in HEK293 cells overexpressing wild-type and mutant SLC30A9 showed lower zinc concentration within mutant rather than wild-type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is through defective function of this novel activity of SLC30A9 rather than by a defect in its previously described role in transcriptional activation of Wnt signalling.

A Look into the Biochemistry of Magnetosome Biosynthesis in Magnetotactic Bacteria.

Magnetosomes are protein-rich membrane organelles that encapsulate magnetite or greigite and whose chain alignment enables magnetotactic bacteria (MTB) to sense the geomagnetic field. As these bacteria synthesize uniform magnetic particles, their biomineralization mechanism is of great interest among researchers from different fields, from material engineering to medicine. Both magnetosome formation and magnetic particle synthesis are highly controlled processes that can be divided into several crucial steps: membrane invagination from the inner-cell membrane, protein sorting, the magnetosomes' arrangement into chains, iron transport, chemical environment regulation of the magnetosome lumen, magnetic particle nucleation, and finally crystal growth, size, and morphology control. This complex system involves an ensemble of unique proteins that participate in different stages during magnetosome formation, some of which were extensively studied in recent years. Here, we present the current knowledge on magnetosome biosynthesis with a focus on the different proteins and the main biochemical pathways along this process.

Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance.

Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins' proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human.

From invagination to navigation: The story of magnetosome-associated proteins in magnetotactic bacteria.

Magnetotactic bacteria (MTB) are a group of Gram-negative microorganisms that are able to sense and change their orientation in accordance with the geomagnetic field. This unique capability is due to the presence of a special suborganelle called the magnetosome, composed of either a magnetite or gregite crystal surrounded by a lipid membrane. MTB were first detected in 1975 and since then numerous efforts have been made to clarify the special mechanism of magnetosome formation at the molecular level. Magnetosome formation can be divided into several steps, beginning with vesicle invagination from the cell membrane, through protein sorting, followed by the combined steps of iron transportation, biomineralization, and the alignment of magnetosomes into a chain. The magnetosome-chain enables the sensing of the magnetic field, and thus, allows the MTB to navigate. It is known that magnetosome formation is tightly controlled by a distinctive set of magnetosome-associated proteins that are encoded mainly in a genomically conserved region within MTB called the magnetosome island (MAI). Most of these proteins were shown to have an impact on the magnetism of MTB. Here, we describe the process in which the magnetosome is formed with an emphasis on the different proteins that participate in each stage of the magnetosome formation scheme.

Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using in vitro TF-DNA preferences obtained from the universal protein binding microarrays (PBM) for ~90 eukaryotic TFs belonging to 22 different DNA-binding domain types. As a result of this new analysis, we conclude that nonconsensus protein-DNA binding is a widespread phenomenon that significantly affects protein-DNA binding preferences and need not require the presence of consensus (specific) TFBSs in order to achieve genome-wide TF-DNA binding specificity.

BtcA, A class IA type III chaperone, interacts with the BteA N-terminal domain through a globular/non-globular mechanism.

Bordetella pertussis, the etiological agent of "whooping cough" disease, utilizes the type III secretion system (T3SS) to deliver a 69 kDa cytotoxic effector protein, BteA, directly into the host cells. As with other T3SS effectors, prior to its secretion BteA binds BtcA, a 13.9 kDa protein predicted to act as a T3SS class IA chaperone. While this interaction had been characterized for such effector-chaperone pairs in other pathogens, it has yet to be fully investigated in Bordetella. Here we provide the first biochemical proof that BtcA is indeed a class IA chaperone, responsible for the binding of BteA's N-terminal domain. We bring forth extensive evidence that BtcA binds its substrate effector through a dual-interface binding mechanism comprising of non-globular and bi-globular interactions at a moderate micromolar level binding affinity. We demonstrate that the non-globular interactions involve the first 31 N-terminal residues of BteA287 and their removal leads to destabilization of the effector-chaperone complex and lower binding affinities to BtcA. These findings represent an important first step towards a molecular understanding of BteA secretion and cell entry.