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CLC channels and transporters have large C-terminal regions which contain two cystathionine β-synthetase (CBS) domains. It has been hypothesized that conformational changes in these domains upon nucleotide binding modulate the gating of the CLC dimer. It is not clear how rearrangements that occur in the CBS domains are transmitted to the ion pathway, as CBS domains interact with the rest of the channel at multiple locations and some of these sites are not visible in recent solved cryogenic electron microscopy structures or are difficult to model using the AlphaFold server. Using ClC-1 as a model, we started working with a described ClC-1 mutation (H835R) located in the first alpha helix of the CBS2 domain which changes the voltage dependence of gating. We then identified several residues located in the disorganized loop after helix R (R-linker) that revert the phenotype of this mutation. We additionally proved that R-linker's function is connected to the CBS2 domain as current intensity, plasma membrane levels and gating defects of several R-linker variants were corrected by adding the mutation H835R. Furthermore, cross-linking studies using newly developed split-cysless ClC-1 channels containing specific cysteine mutants in the R-linker and the CBS2 domain indicate that these two regions are in close contact. Considering these new results, we propose that conformational changes occurring in the CBS domains could be transmitted to the CLC intracellular chloride binding site by means of its interaction with the R-linker. KEY POINTS: CBS domains, which are present as pairs in CLC proteins, are involved in the regulation by nucleotides of CLC gating. It is not clear how CBS domains interact with different regions of the transmembrane (TM) region to regulate gating. Using ClC-1 as a model, we investigated how a mutation in the second CBS domain dramatically changes the voltage dependence of gating taking advantage of recently solved structures and AlphaFold models of CLC proteins. Thus, we identified in the linker after helix R (R-linker) several revertant mutations of the gating defects caused by a mutation in the second CBS2 domain and vice versa, indicating that these two regions functionally interact. These interactions were biochemically proven by employing cysteine cross-linkings using a newly developed split-cysless ClC-1 channel. Based on these findings we experimentally prove that the R-linker is a key element for the transmission of the CBS conformational rearrangements to the TM region., (© 2025 The Authors. The Journal of Physiology © 2025 The Physiological Society.)

Background: Ashbya gossypii is a filamentous fungus widely utilized for industrial riboflavin production and has a great potential as a microbial chassis for synthesizing other valuable metabolites such as folates, biolipids, and limonene. Engineered strains of A. gossypii can effectively use various waste streams, including xylose-rich feedstocks. Notably, A. gossypii has been identified as a proficient biocatalyst for producing limonene from xylose-rich sources. This study aims to investigate the capability of engineered A. gossypii strains to produce various plant monoterpenes using agro-industrial waste as carbon sources., Results: We overexpressed heterologous terpene synthases to produce acyclic, monocyclic, and bicyclic monoterpenes in two genetic backgrounds of A. gossypii. These backgrounds included an NPP synthase orthogonal pathway and a mutant erg20 F95W allele with reduced FPP synthase activity. Our findings demonstrate that A. gossypii can synthesize linalool, limonene, pinene, and sabinene, with terpene synthases showing differential substrate selectivity for NPP or GPP precursors. Additionally, co-overexpression of endogenous HMG1 and ERG12 with heterologous NPP synthase and terpene synthases significantly increased sabinene yields from xylose-containing media. Using mixed formulations of corn-cob lignocellulosic hydrolysates and either sugarcane or beet molasses, we achieved limonene and sabinene productions of 383 mg/L and 684.5 mg/L, respectively, the latter representing a significant improvement compared to other organisms in flask culture mode., Conclusions: Engineered A. gossypii strains serve as a suitable platform for assessing plant terpene synthase functionality and substrate selectivity in vivo, which are crucial to understand monoterpene bioproduction. The NPP synthase pathway markedly enhances limonene and sabinene production in A. gossypii, achieving levels comparable to those of other industrial microbial producers. Furthermore, these engineered strains offer a novel approach for producing monoterpenes through the valorization of agro-industrial wastes., (© 2024. The Author(s).)

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased., (© 2024 Calise et al.)

Importance: Small proteins containing fewer than 70 amino acids, which were previously disregarded due to computational prediction and biochemical detection challenges, have gained increased attention in the scientific community in recent years. However, the number of functionally characterized small proteins, especially in archaea, is still limited. Here, by using biochemical and genetic approaches, we demonstrate a crucial role of the small protein sP36 in the nitrogen metabolism of M. mazei , which modulates the ammonium transporter AmtB1 according to nitrogen availability. This modulation might represent an ancient archaeal mechanism of AmtB1 inhibition, in contrast to the well-studied uridylylation-dependent regulation in bacteria., Competing Interests: The authors declare no conflict of interest.

IMP dehydrogenase and GMP reductase are enzymes from the same protein family with analogous structures and catalytic mechanisms that have gained attention because of their essential roles in nucleotide metabolism and as potential drug targets. This study focusses on GuaB3, a less-explored enzyme within this family. Phylogenetic analysis uncovers GuaB3's independent evolution from other members of the family and it predominantly occurs in Cyanobacteria. Within this group, GuaB3 functions as a unique IMP dehydrogenase, while its counterpart in Actinobacteria has a yet unknown function. Synechocystis sp. PCC6803 GuaB3 structures demonstrate differences in the active site compared to canonical IMP dehydrogenases, despite shared catalytic mechanisms. These findings highlight the essential role of GuaB3 in Cyanobacteria, provide insights into the diversity and evolution of the IMP dehydrogenase protein family, and reveal a distinctive characteristic in nucleotide metabolism, potentially aiding in combating harmful cyanobacterial blooms-a growing concern for humans and wildlife., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the high activity assembly interface, still allowing assembly of low activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands., Competing Interests: Competing Interest Statement: The authors declare that they have no competing interests.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is an evolutionarily conserved enzyme that mediates the first committed step in de novo guanine nucleotide biosynthetic pathway. It is an essential enzyme in purine nucleotide biosynthesis that modulates the metabolic flux at the branch point between adenine and guanine nucleotides. IMPDH plays key roles in cell homeostasis, proliferation, and the immune response, and is the cellular target of several drugs that are widely used for antiviral and immunosuppressive chemotherapy. IMPDH enzyme is tightly regulated at multiple levels, from transcriptional control to allosteric modulation, enzyme filamentation, and posttranslational modifications. Herein, we review recent developments in our understanding of the mechanisms of IMPDH regulation, including all layers of allosteric control that fine-tune the enzyme activity., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

IMP dehydrogenase(IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide pathway. In eukaryotic cells, GTP binding to the regulatory domain allosterically controls the activity of IMPDH by a mechanism that is fine-tuned by post-translational modifications and enzyme polymerization. Nonetheless, the mechanisms of regulation of IMPDH in bacterial cells remain unclear. Using biochemical, structural, and evolutionary analyses, we demonstrate that, in most bacterial phyla, (p)ppGpp compete with ATP to allosterically modulate IMPDH activity by binding to a, previously unrecognized, conserved high affinity pocket within the regulatory domain. This pocket was lost during the evolution of Proteobacteria, making their IMPDHs insensitive to these alarmones. Instead, most proteobacterial IMPDHs evolved to be directly modulated by the balance between ATP and GTP that compete for the same allosteric binding site. Altogether, we demonstrate that the activity of bacterial IMPDHs is allosterically modulated by a universally conserved nucleotide-controlled conformational switch that has divergently evolved to adapt to the specific particularities of each organism. These results reconcile the reported data on the crosstalk between (p)ppGpp signaling and the guanine nucleotide biosynthetic pathway and reinforce the essential role of IMPDH allosteric regulation on bacterial GTP homeostasis., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

We have structurally and functionally characterized Skl and Pal endolysins, the latter being the first endolysin shown to kill effectively Streptococcus pneumoniae , a leading cause of deathly diseases. We have proved that Skl and Pal are cysteine-amidases whose catalytic domains, from CHAP and Amidase_5 families, respectively, share an α 3 β 6 -fold with papain-like topology. Catalytic triads are identified (for the first time in Amidase_5 family), and residues relevant for substrate binding and catalysis inferred from in silico models, including a calcium-binding site accounting for Skl dependence on this cation for activity. Both endolysins contain a choline-binding domain (CBD) with a β-solenoid fold (homology modeled) and six conserved choline-binding loci whose saturation induced dimerization. Remarkably, Pal and Skl dimers display a common overall architecture, preserved in choline-bound dimers of pneumococcal lysins with other catalytic domains and bond specificities, as disclosed using small angle X-ray scattering (SAXS). Additionally, Skl is proved to be an efficient anti-pneumococcal agent that kills multi-resistant strains and clinical emergent-serotype isolates. Interestingly, Skl and Pal time-courses of pneumococcal lysis were sigmoidal, which might denote a limited access of both endolysins to target bonds at first stages of lysis. Furthermore, their DTT-mediated activation, of relevance for other cysteine-peptidases, cannot be solely ascribed to reversal of catalytic-cysteine oxidation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gallego-Páramo, Hernández-Ortiz, Buey, Rico-Lastres, García, Díaz, García and Menéndez.)

Thioredoxin reductases control the redox state of thioredoxins (Trxs)-ubiquitous proteins that regulate a spectrum of enzymes by dithiol-disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here, we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox-sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

We report the in vivo regulation of Inosine-5´-monophosphate dehydrogenase 1 (IMPDH1) in the retina. IMPDH1 catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, impacting the cellular pools of GMP, GDP and GTP. Guanine nucleotide homeostasis is central to photoreceptor cells, where cGMP is the signal transducing molecule in the light response. Mutations in IMPDH1 lead to inherited blindness. We unveil a light-dependent phosphorylation of retinal IMPDH1 at Thr 159 /Ser 160 in the Bateman domain that desensitizes the enzyme to allosteric inhibition by GDP/GTP. When exposed to bright light, living mice increase the rate of GTP and ATP synthesis in their retinas; concomitant with IMPDH1 aggregate formation at the outer segment layer. Inhibiting IMPDH activity in living mice delays rod mass recovery. We unveil a novel mechanism of regulation of IMPDH1 in vivo, important for understanding GTP homeostasis in the retina and the pathogenesis of adRP10 IMPDH1 mutations., Competing Interests: AP, SL, DF, AJ, AS, JA, PL, JR, EM, IC, OY, Pd, RB, AM No competing interests declared, (© 2020, Plana-Bonamaisó et al.)

Microtubules (MTs) are hollow cylinders made of tubulin, a GTPase responsible for essential functions during cell growth and division, and thus, key target for anti-tumor drugs. In MTs, GTP hydrolysis triggers structural changes in the lattice, which are responsible for interaction with regulatory factors. The stabilizing GTP-cap is a hallmark of MTs and the mechanism of the chemical-structural link between the GTP hydrolysis site and the MT lattice is a matter of debate. We have analyzed the structure of tubulin and MTs assembled in the presence of fluoride salts that mimic the GTP-bound and GDP•P i transition states. Our results challenge current models because tubulin does not change axial length upon GTP hydrolysis. Moreover, analysis of the structure of MTs assembled in the presence of several nucleotide analogues and of taxol allows us to propose that previously described lattice expansion could be a post-hydrolysis stage involved in P i release., Competing Interests: JE, FJ, SK, RB, FB, AP, DL, CK, TY, HI, LD, IB, MS, DC, SK, JD, MO No competing interests declared, (© 2020, Estévez-Gallego et al.)

The filamentous fungus Ashbya gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. gossypii to grow using low-cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. gossypii of other high-added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one-vector CRISPR/Cas9 system for genomic engineering of A. gossypii. We demonstrate the efficiency of the system as a marker-less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker-less manner., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the de novo guanine nucleotide biosynthetic pathway. Because of its involvement in the control of cell division and proliferation, IMPDH represents a therapeutic for managing several diseases, including microbial infections and cancer. IMPDH must be tightly regulated, but the molecular mechanisms responsible for its physiological regulation remain unknown. To this end, we recently reported an important role of adenine and guanine mononucleotides that bind to the regulatory Bateman domain to allosterically modulate the catalytic activity of eukaryotic IMPDHs. Here, we have used enzyme kinetics, X-ray crystallography, and small-angle X-ray scattering (SAXS) methodologies to demonstrate that adenine/guanine dinucleoside polyphosphates bind to the Bateman domain of IMPDH from the fungus Ashbya gossypii with submicromolar affinities. We found that these dinucleoside polyphosphates modulate the catalytic activity of IMPDHs in vitro by efficiently competing with the adenine/guanine mononucleotides for the allosteric sites. These results suggest that dinucleoside polyphosphates play important physiological roles in the allosteric regulation of IMPDHs by adding an additional mechanism for fine-tuning the activities of these enzymes. We propose that these findings may have important implications for the design of therapeutic strategies to inhibit IMPDHs., (© 2019 Fernández-Justel et al.)

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo GTP biosynthetic pathway and plays essential roles in cell proliferation. As a clinical target, IMPDH has been studied for decades, but it has only been within the last years that we are starting to understand the complexity of the mechanisms of its physiological regulation. Here, we report structural and functional insights into how adenine and guanine nucleotides control a conformational switch that modulates the assembly of the two human IMPDH enzymes into cytoophidia and allosterically regulates their catalytic activity. In vitro reconstituted micron-length cytoophidia-like structures show catalytic activity comparable to unassembled IMPDH but, in turn, are more resistant to GTP/GDP allosteric inhibition. Therefore, IMPDH cytoophidia formation facilitates the accumulation of high levels of guanine nucleotides when the cell requires it. Finally, we demonstrate that most of the IMPDH retinopathy-associated mutations abrogate GTP/GDP-induced allosteric inhibition and alter cytoophidia dynamics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Ferredoxin-dependent thioredoxin reductase was identified 35 y ago in the fermentative bacterium Clostridium pasteurianum [Hammel KE, Cornwell KL, Buchanan BB (1983) Proc Natl Acad Sci USA 80:3681-3685]. The enzyme, a flavoprotein, was strictly dependent on ferredoxin as reductant and was inactive with either NADPH or NADH. This early work has not been further pursued. We have recently reinvestigated the problem and confirmed that the enzyme, here designated ferredoxin-dependent flavin thioredoxin reductase (FFTR), is a flavoprotein. The enzyme differs from ferredoxin-thioredoxin reductase (FTR), which has a signature [4Fe-4S] cluster, but shows structural similarities to NADP-dependent thioredoxin reductase (NTR). Comparative amino acid sequence analysis showed that FFTR is present in a number of clostridial species, some of which lack both FTR and an archetypal NTR. We have isolated, crystallized, and determined the structural properties of FFTR from a member of this group, Clostridium acetobutylicum , both alone and in complex with Trx. The structures showed an elongated FFTR homodimer, each monomer comprising two Rossmann domains and a noncovalently bound FAD cofactor that exposes the isoalloxazine ring to the solvent. The FFTR structures revealed an alternative domain organization compared with NTR that enables the enzyme to accommodate Fdx rather than NADPH. The results suggest that FFTR exists in a range of conformations with varying degrees of domain separation in solution and that the stacking between the two redox-active groups for the transfer of reducing equivalents results in a profound structural reorganization. A mechanism in accord with the findings is proposed., Competing Interests: Conflict of interest statement: B.B.B. and I.A.B. are coauthors of a 2017 retrospective article.

The redox regulation of proteins via reversible dithiol/disulfide exchange reactions involves the thioredoxin system, which is composed of a reductant, a thioredoxin reductase (TR), and thioredoxin (Trx). In the pyridine nucleotide-dependent Trx reduction pathway, reducing equivalents, typically from reduced nicotinamide adenine dinucleotide phosphate (NADPH), are transferred from NADPH-TR (NTR) to Trx and, in turn, to target proteins, thus resulting in the reversible modification of the structural and functional properties of the targets. NTR enzymes contain three functional sites: an NADPH binding pocket, a non-covalently bound flavin cofactor, and a redox-active disulfide in the form of CxxC. With the aim of increasing our knowledge of the thioredoxin system in archaea, we here report the high-resolution crystal structure of NTR from the methane-generating organism Methanosarcina mazei strain Gö1 (MmNTR) at 2.6 Å resolution. Based on the crystals presently described, MmNTR assumes an overall fold that is nearly identical to the archetypal fold of authentic NTRs; however, surprisingly, we observed no electron density for flavin adenine dinucleotide (FAD) despite the well-defined and conserved FAD-binding cavity in the folded module. Remarkably, the dimers of the apo-protein within the crystal were different from those observed by small angle X-ray scattering (SAXS) for the holo-protein, suggesting that the binding of the flavin cofactor does not require major protein structural rearrangements. Rather, binding results in the stabilization of essential parts of the structure, such as those involved in dimer stabilization. Altogether, this structure represents the example of an apo-form of an NTR that yields important insight into the effects of the cofactor on protein folding.

Endolysins, the cell wall lytic enzymes encoded by bacteriophages to release the phage progeny, are among the top alternatives to fight against multiresistant pathogenic bacteria; one of the current biggest challenges to global health. Their narrow range of susceptible bacteria relies, primarily, on targeting specific cell-wall receptors through specialized modules. The cell wall-binding domain of Cpl-7 endolysin, made of three CW_7 repeats, accounts for its extended-range of substrates. Using as model system the cell wall-binding domain of Cpl-7, here we describe the molecular basis for the bacterial cell wall recognition by the CW_7 motif, which is widely represented in sequences of cell wall hydrolases. We report the crystal and solution structure of the full-length domain, identify N-acetyl-D-glucosaminyl-(β1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) as the peptidoglycan (PG) target recognized by the CW_7 motifs, and characterize feasible GMDP-CW_7 contacts. Our data suggest that Cpl-7 cell wall-binding domain might simultaneously bind to three PG chains, and also highlight the potential use of CW_7-containing lysins as novel anti-infectives.

Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications., Competing Interests: The authors declare no conflict of interest.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for nucleotide metabolism and cell proliferation. Despite IMPDH is the target of drugs with antiviral, immunosuppressive and antitumor activities, its physiological mechanisms of regulation remain largely unknown. Using the enzyme from the industrial fungus Ashbya gossypii, we demonstrate that the binding of adenine and guanine nucleotides to the canonical nucleotide binding sites of the regulatory Bateman domain induces different enzyme conformations with significantly distinct catalytic activities. Thereby, the comparison of their high-resolution structures defines the mechanistic and structural details of a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity of eukaryotic IMPDHs. Remarkably, retinopathy-associated mutations lie within the mechanical hinges of the conformational change, highlighting its physiological relevance. Our results expand the mechanistic repertoire of Bateman domains and pave the road to new approaches targeting IMPDHs.

Riboflavin (vitamin B 2 ) is an essential nutrient for humans and animals that must be obtained from the diet. To ensure an optimal supply, riboflavin is used on a large scale as additive in the food and feed industries. Here, we describe a historical overview of the industrial process of riboflavin production starting from its discovery and the need to produce the vitamin in bulk at prices that would allow for their use in human and animal nutrition. Riboflavin was produced industrially by chemical synthesis for many decades. At present, the development of economical and eco-efficient fermentation processes, which are mainly based on Bacillus subtilis and Ashbya gossypii strains, has replaced the synthetic process at industrial scale. A detailed account is given of the development of the riboflavin overproducer strains as well as future prospects for its improvement.

Ashbya gossypii is a filamentous fungus that naturally overproduces riboflavin, and it is currently exploited for the industrial production of this vitamin. The utilization of A. gossypii for biotechnological applications presents important advantages such as the utilization of low-cost culture media, inexpensive downstream processing and a wide range of molecular tools for genetic manipulation, thus making A. gossypii a valuable biotechnological chassis for metabolic engineering. A. gossypii has been shown to accumulate high levels of lipids in oil-based culture media; however, the lipid biosynthesis capacity is rather limited when grown in sugar-based culture media. In this study, by altering the fatty acyl-CoA pool and manipulating the regulation of the main ∆9 desaturase gene, we have obtained A. gossypii strains with significantly increased (up to fourfold) de novo lipid biosynthesis using glucose as the only carbon source in the fermentation broth. Moreover, these strains were efficient biocatalysts for the conversion of carbohydrates from sugarcane molasses to biolipids, able to accumulate lipids up to 25% of its cell dry weight. Our results represent a proof of principle showing the promising potential of A. gossypii as a competitive microorganism for industrial biolipid production using cost-effective feed stocks., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Background: Ashbya gossypii is a filamentous fungus that is currently exploited for the industrial production of riboflavin. The utilization of A. gossypii as a microbial biocatalyst is further supported by its ability to grow in low-cost feedstocks, inexpensive downstream processing and the availability of an ease to use molecular toolbox for genetic and genomic modifications. Consequently, A. gossypii has been also introduced as an ideal biotechnological chassis for the production of inosine, folic acid, and microbial oils. However, A. gossypii cannot use xylose, the most common pentose in hydrolysates of plant biomass., Results: In this work, we aimed at designing A. gossypii strains able to utilize xylose as the carbon source for the production of biolipids. An endogenous xylose utilization pathway was identified and overexpressed, resulting in an A. gossypii xylose-metabolizing strain showing prominent conversion rates of xylose to xylitol (up to 97% after 48 h). In addition, metabolic flux channeling from xylulose-5-phosphate to acetyl-CoA, using aheterologous phosphoketolase pathway, increased the lipid content in the xylose-metabolizing strain a 54% over the parental strain growing in glucose-based media. This increase raised to 69% when lipid accumulation was further boosted by blocking the beta-oxidation pathway., Conclusions: Ashbya gossypii has been engineered for the utilization of xylose. We present here a proof-of-concept study for the production of microbial oils from xylose in A. gossypii , thus introducing a novel biocatalyst with very promising properties in developing consolidated bioprocessing to produce fine chemicals and biofuels from xylose-rich hydrolysates of plant biomass.

EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice. Microtubules assembled in the presence of GTP, different GTP analogues or cell extracts display similarly curved sheets at their growing ends, which gradually straighten as their protofilament number increases until they close into a tube. Together, our data provide unique structural information on the interaction of EB1 with growing microtubule ends. They further offer insights into the conformational changes that tubulin dimers undergo during microtubule assembly and the architecture of the GTP-cap region.

Plakins are large multi-domain proteins that interconnect cytoskeletal structures. Plectin is a prototypical plakin that tethers intermediate filaments to membrane-associated complexes. Most plakins contain a plakin domain formed by up to nine spectrin repeats (SR1-SR9) and an SH3 domain. The plakin domains of plectin and other plakins harbor binding sites for junctional proteins. We have combined x-ray crystallography with small angle x-ray scattering (SAXS) to elucidate the structure of the plakin domain of plectin, extending our previous analysis of the SR1 to SR5 region. Two crystal structures of the SR5-SR6 region allowed us to characterize its uniquely wide inter-repeat conformational variability. We also report the crystal structures of the SR7-SR8 region, refined to 1.8 Å, and the SR7-SR9 at lower resolution. The SR7-SR9 region, which is conserved in all other plakin domains, forms a rigid segment stabilized by uniquely extensive inter-repeat contacts mediated by unusually long helices in SR8 and SR9. Using SAXS we show that in solution the SR3-SR6 and SR7-SR9 regions are rod-like segments and that SR3-SR9 of plectin has an extended shape with a small central kink. Other plakins, such as bullous pemphigoid antigen 1 and microtubule and actin cross-linking factor 1, are likely to have similar extended plakin domains. In contrast, desmoplakin has a two-segment structure with a central flexible hinge. The continuous versus segmented structures of the plakin domains of plectin and desmoplakin give insight into how different plakins might respond to tension and transmit mechanical signals., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Inosine is a nucleoside with growing biotechnological interest due to its recently attributed beneficial health effects and as a convenient precursor of the umami flavor. At present, most of the industrial inosine production relies on bacterial fermentations. In this work, we have metabolically engineered the filamentous fungus Ashbya gossypii to obtain strains able to excrete high amounts of inosine to the culture medium. We report that the disruption of only two key genes of the purine biosynthetic pathway efficiently redirect the metabolic flux, increasing 200-fold the excretion of inosine with respect to the wild type, up to 2.2 g/L. These results allow us to propose A. gossypii as a convenient candidate for large-scale nucleoside production, especially in view of the several advantages that Ashbya has with respect to the bacterial systems used at present for the industrial production of this food additive. Biotechnol. Bioeng. 2016;113: 2060-2063. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Vitamins and related compounds, such as provitamins, biopigments and antioxidants, belong to those few chemicals that appeal in a positive way to most people. These terms sound synonymous to vitality, good health and mental strenght, even to the layman. Everyone of us needs his/her daily intake of (pro)vitamins and antioxidants, normally provided by a balanced and varied diet. However, current food habits or preferences, food availabilities, as well as food processing, preservation or cooking methodologies and technologies, do not always assure a sufficient balanced natural daily (pro)vitamin supply to a healthy individual, and even more so for a stressed or sick human being. Today, modern society is seldom confronted with the notorious avitaminoses of the past, well known to the Western World, but they do still occur frequently in overpopulated, war-ridden, poverty- or famine-struck regions on our globe, as well as for surprisingly large population groups in developed countries., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Plectin and BPAG1e belong to the plakin family of high-molecular-weight proteins that interconnect the cytoskeletal systems and anchor them to junctional complexes. Plectin and BPAG1e are prototypical plakins with a similar tripartite modular structure. The N- and C-terminal regions are built of multiple discrete structural domains, while the central rod domain mediates dimerization by coiled-coil interactions. Owing to the mosaic organization of plakins, the structure of their constituent individual domains or small multi-domain segments can be analyzed isolated. Yet, understanding the integrated function of large regions, oligomers, and heterocomplexes of plakins is difficult due to the large and segmented structure. Here, we describe methods for the production of plectin and BPAG1e samples suitable for structural and biophysical analysis. In addition, we discuss the combination of hybrid methods that yield information at several resolution levels to study the complex, multi-domain, and flexible structure of plakins., (© 2016 Elsevier Inc. All rights reserved.)

Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the ratelimiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches. Herein, we report the functional and structural characterization of the IMPDH enzyme from the industrial fungus Ashbya gossypii. Our data show that the overexpression of the IMPDH gene increases the metabolic flux through the guanine pathway and ultimately enhances 40 % riboflavin production with respect to the wild type. Also, IMPDH disruption results in a 100-fold increase of inosine excretion to the culture media. Our results contribute to the developing metabolic engineering toolbox aiming at improving the production of metabolites with biotechnological interest in A. gossypii.

Background: Inosine and guanosine monophosphate nucleotides are convenient sources of the umami flavor, with attributed beneficial health effects that have renewed commercial interest in nucleotide fermentations. Accordingly, several bacterial strains that excrete high levels of inosine and guanosine nucleosides are currently used in the food industry for this purpose., Results: In the present study, we show that the filamentous fungus Ashbya gossypii, a natural riboflavin overproducer, excretes high amounts of inosine and guanosine nucleosides to the culture medium. Following a rational metabolic engineering approach of the de novo purine nucleotide biosynthetic pathway, we increased the excreted levels of inosine up to 27-fold., Conclusions: We generated Ashbya gossypii strains with improved production titers of inosine and guanosine. Our results point to Ashbya gossypii as the first eukaryotic microorganism representing a promising candidate, susceptible to further manipulation, for industrial nucleoside fermentation.

Integrin α6β4 is a major component of hemidesmosomes that mediate the stable anchorage of epithelial cells to the underlying basement membrane. Integrin α6β4 has also been implicated in cell proliferation and migration and in carcinoma progression. The third and fourth fibronectin type III domains (FnIII-3,4) of integrin β4 mediate binding to the hemidesmosomal proteins BPAG1e and BPAG2, and participate in signalling. Here, it is demonstrated that X-ray crystallography, small-angle X-ray scattering and double electron-electron resonance (DEER) complement each other to solve the structure of the FnIII-3,4 region. The crystal structures of the individual FnIII-3 and FnIII-4 domains were solved and the relative arrangement of the FnIII domains was elucidated by combining DEER with site-directed spin labelling. Multiple structures of the interdomain linker were modelled by Monte Carlo methods complying with DEER constraints, and the final structures were selected against experimental scattering data. FnIII-3,4 has a compact and cambered flat structure with an evolutionary conserved surface that is likely to correspond to a protein-interaction site. Finally, this hybrid method is of general application for the study of other macromolecules and complexes.

Microtubule-targeting agents are widely used for the treatment of cancer and as tool compounds to study the microtubule cytoskeleton. BAL27862 is a novel microtubule-destabilizing drug that is currently undergoing phase I clinical evaluation as the prodrug BAL101553. The drug is a potent inhibitor of tumor cell growth and shows a promising activity profile in a panel of human cancer models resistant to clinically relevant microtubule-targeting agents. Here, we evaluated the molecular mechanism of the tubulin-BAL27862 interaction using a combination of cell biology, biochemistry and structural biology methods. Tubulin-binding assays revealed that BAL27862 potently inhibited tubulin assembly at 37 °C with an IC50 of 1.4 μM and bound to unassembled tubulin with a stoichiometry of 1 mol/mol tubulin and a dissociation constant of 244±30 nM. BAL27862 bound to tubulin independently of vinblastine, without the formation of tubulin oligomers. The kinetics of BAL27862 binding to tubulin were distinct from those of colchicine, with evidence of competition between BAL27862 and colchicine for binding. Determination of the tubulin-BAL27862 structure by X-ray crystallography demonstrated that BAL27862 binds to the same site as colchicine at the intradimer interface. Comparison of crystal structures of tubulin-BAL27862 and tubulin-colchicine complexes shows that the binding mode of BAL27862 to tubulin is similar to that of colchicine. However, comparative analyses of the effects of BAL27862 and colchicine on the microtubule mitotic spindle and in tubulin protease-protection experiments suggest different outcomes of tubulin binding. Taken together, our data define BAL27862 as a potent, colchicine site-binding, microtubule-destabilizing agent with distinct effects on microtubule organization., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We describe an algorithm for phasing protein crystal X-ray diffraction data that identifies, retrieves, refines and exploits general tertiary structural information from small fragments available in the Protein Data Bank. The algorithm successfully phased, through unspecific molecular replacement combined with density modification, all-helical, mixed alpha-beta, and all-beta protein structures. The method is available as a software implementation: Borges.

Microtubule plus-end-tracking proteins (+TIPs) specifically localize to the growing plus-ends of microtubules to regulate microtubule dynamics and functions. A large group of +TIPs contain a short linear motif, SXIP, which is essential for them to bind to end-binding proteins (EBs) and target microtubule ends. The SXIP sequence site thus acts as a widespread microtubule tip localization signal (MtLS). Here we have analyzed the sequence-function relationship of a canonical MtLS. Using synthetic peptide arrays on membrane supports, we identified the residue preferences at each amino acid position of the SXIP motif and its surrounding sequence with respect to EB binding. We further developed an assay based on fluorescence polarization to assess the mechanism of the EB-SXIP interaction and to correlate EB binding and microtubule tip tracking of MtLS sequences from different +TIPs. Finally, we investigated the role of phosphorylation in regulating the EB-SXIP interaction. Together, our results define the sequence determinants of a canonical MtLS and provide the experimental data for bioinformatics approaches to carry out genome-wide predictions of novel +TIPs in multiple organisms.

End binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in the form of comet tails and regulate microtubule dynamics. However, the mechanism by which they regulate microtubule dynamics is not well understood. Using full-length EB1 and a minimal functional fragment of CLIP-170 (ClipCG12), we found that EB1 and CLIP-170 cooperatively regulate microtubule dynamic instability at concentrations below which neither protein is effective. By use of small-angle X-ray scattering and analytical ultracentrifugation, we found that ClipCG12 adopts a largely extended conformation with two noninteracting CAP-Gly domains and that it formed a complex in solution with EB1. Using a reconstituted steady-state mammalian microtubule system, we found that at a low concentration of 250 nM, neither EB1 nor ClipCG12 individually modulated plus-end dynamic instability. Higher concentrations (up to 2 μM) of the two proteins individually did modulate dynamic instability, perhaps by a combination of effects at the tips and along the microtubule lengths. However, when low concentrations (250 nM) of EB1 and ClipCG12 were present together, the mixture modulated dynamic instability considerably. Using a pulsing strategy with [γ(32)P]GTP, we further found that unlike EB1 or ClipCG12 alone, the EB1-ClipCG12 mixture partially depleted the microtubule ends of stably bound (32)P(i). Together, our results suggest that EB1 and ClipCG12 act cooperatively to regulate microtubule dynamics. They further indicate that stabilization of microtubule plus ends by the EB1-ClipCG12 mixture may involve modification of an aspect of the stabilizing cap., (© 2012 American Chemical Society)

End-binding proteins (EBs) comprise a conserved family of microtubule plus end-tracking proteins. The concerted action of calponin homology (CH), linker, and C-terminal domains of EBs is important for their autonomous microtubule tip tracking, regulation of microtubule dynamics, and recruitment of numerous partners to microtubule ends. Here we report the detailed structural and biochemical analysis of mammalian EBs. Small-angle X-ray scattering, electron microscopy, and chemical cross-linking in combination with mass spectrometry indicate that EBs are elongated molecules with two interacting CH domains, an arrangement reminiscent of that seen in other microtubule- and actin-binding proteins. Removal of the negatively charged C-terminal tail did not affect the overall conformation of EBs; however, it increased the dwell times of EBs on the microtubule lattice in microtubule tip-tracking reconstitution experiments. An even more stable association with the microtubule lattice was observed when the entire negatively charged C-terminal domain of EBs was replaced by a neutral coiled-coil motif. In contrast, the interaction of EBs with growing microtubule tips was not significantly affected by these C-terminal domain mutations. Our data indicate that long-range electrostatic repulsive interactions between the C-terminus and the microtubule lattice drive the specificity of EBs for growing microtubule ends.

Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of spectrin repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 Å resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudo-binding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in spectrins. The flexibility of the plakin domain, in analogy with spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.

The oxaloacetate decarboxylase primary Na(+) pump (OAD) is an essential membrane protein complex that functions in the citrate fermentation pathway of some pathogenic bacteria under anaerobic conditions. OAD contains three different subunits: Oad-α, a biotinylated extrinsic protein that catalyzes the α-ketodecarboxylation of oxaloacetate; Oad-γ, a structural bitopic membrane protein whose cytosolic tail (named as Oad-γ') binds tightly to Oad-α; and Oad-β, a multispan transmembrane α-helical protein that constitutes the Na(+) channel. How OAD is organized structurally at the membrane and what the molecular determinants are that lead to an efficient energy coupling mechanism remain elusive. In the present work, we elucidate the stoichiometry of the native complex as well as the low resolution structure of the peripheral components of OAD (Oad-α and Oad-γ') by small angle x-ray scattering. Our results point to a quaternary assembly similar to the pyruvate carboxylase complex organization. Herein, we propose a model in which the association in pairs of Oad-α dimers, mediated by Oad-γ, results in the acquisition of a functional oligomeric state at the bacterial membrane. New structural insights for the conformational rearrangements associated with the carboxylbiotin transfer reaction within OAD are provided.

The kinesin-13 family member mitotic centromere-associated kinesin (MCAK) is a potent microtubule depolymerase. Paradoxically, in cells it accumulates at the growing, rather than the shortening, microtubule plus ends. This plus-end tracking behavior requires the interaction between MCAK and members of the end-binding protein (EB) family, but the effect of EBs on the microtubule-destabilizing activity of MCAK and the functional significance of MCAK accumulation at the growing microtubule tips have so far remained elusive. Here, we dissect the functional interplay between MCAK and EB3 by reconstituting EB3-dependent MCAK activity on dynamic microtubules in vitro. Whereas MCAK alone efficiently blocks microtubule assembly, the addition of EB3 restores robust microtubule growth, an effect that is not dependent on the binding of MCAK to EB3. At the same time, EB3 targets MCAK to growing microtubule ends by increasing its association rate with microtubule tips, a process that requires direct interaction between the two proteins. This EB3-dependent microtubule plus-end accumulation does not affect the velocity of microtubule growth or shortening but enhances the capacity of MCAK to induce catastrophes. The combination of MCAK and EB3 thus promotes rapid switching between microtubule growth and shortening, which can be important for remodeling of the microtubule cytoskeleton., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Peloruside is a microtubule-stabilizing agent that targets the same site as laulimalide. It binds to microtubules with a 1:1 stoichiometry and with a binding affinity in the low-muM range; thereby reducing the number of microtubular protofilaments in the same way as paclitaxel. Although the binding affinity of the compound is comparable to that of the low-affinity stabilizing agent sarcodictyin, peloruside is more active in inducing microtubule assembly and is more cytotoxic to tumor cells; this suggests that the peloruside site is a more effective site for stabilizing microtubules. Acetylation of the C24 hydroxyl group results in inactive compounds. According to molecular modeling, this substitution at the C24 hydroxyl group presumably disrupts the interaction of the side chain with Arg320 in the putative binding site on alpha-tubulin. The binding epitope of peloruside on microtubules has been studied by using NMR spectroscopic techniques, and is compatible with the same binding site.

The molecular switch for nucleotide-regulated assembly and disassembly of the main prokaryotic cell division protein FtsZ is unknown despite the numerous crystal structures that are available. We have characterized the functional motions in FtsZ with a computational consensus of essential dynamics, structural comparisons, sequence conservation, and networks of co-evolving residues. Employing this information, we have constructed 17 mutants, which alter the FtsZ functional cycle at different stages, to modify FtsZ flexibility. The mutant phenotypes ranged from benign to total inactivation and included increased GTPase, reduced assembly, and stabilized assembly. Six mutations clustering at the long cleft between the C-terminal beta-sheet and core helix H7 deviated FtsZ assembly into curved filaments with inhibited GTPase, which still polymerize cooperatively. These mutations may perturb the predicted closure of the C-terminal domain onto H7 required for switching between curved and straight association modes and for GTPase activation. By mapping the FtsZ assembly switch, this work also gives insight into FtsZ druggability because the curved mutations delineate the putative binding site of the promising antibacterial FtsZ inhibitor PC190723.

Microtubule plus-end tracking proteins (+TIPs) are involved in many microtubule-based processes. End binding (EB) proteins constitute a highly conserved family of +TIPs. They play a pivotal role in regulating microtubule dynamics and in the recruitment of diverse +TIPs to growing microtubule plus ends. Here we used a combination of methods to investigate the dimerization properties of the three human EB proteins EB1, EB2, and EB3. Based on Förster resonance energy transfer, we demonstrate that the C-terminal dimerization domains of EBs (EBc) can readily exchange their chains in solution. We further document that EB1c and EB3c preferentially form heterodimers, whereas EB2c does not participate significantly in the formation of heterotypic complexes. Measurements of the reaction thermodynamics and kinetics, homology modeling, and mutagenesis provide details of the molecular determinants of homo- versus heterodimer formation of EBc domains. Fluorescence spectroscopy and nuclear magnetic resonance studies in the presence of the cytoskeleton-associated protein-glycine-rich domains of either CLIP-170 or p150(glued) or of a fragment derived from the adenomatous polyposis coli tumor suppressor protein show that chain exchange of EBc domains can be controlled by binding partners. Extension of these studies of the EBc domains to full-length EBs demonstrate that heterodimer formation between EB1 and EB3, but not between EB2 and the other two EBs, occurs both in vitro and in cells as revealed by live cell imaging. Together, our data provide molecular insights for rationalizing the dominant negative control by C-terminal EB domains and form a basis for understanding the functional role of heterotypic chain exchange by EBs in cells.

Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.

Background Information: Hsp90 (90 kDa heat-shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide-binding-dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue)., Results and Discussion: Here, we present the first nucleotide-free structures of the entire eukaryotic Hsp90 (apo-Hsp90) obtained by small-angle X-ray scattering and single-particle cryo-EM (cryo-electron microscopy). We show that, in solution, apo-Hsp90 is in a conformational equilibrium between two open states that have never been described previously. By comparing our cryo-EM maps with HtpG and known Hsp90 structures, we establish that the structural changes involved in switching between the two Hsp90 apo-forms require large movements of the NTD (N-terminal domain) and MD (middle domain) around two flexible hinge regions., Conclusions: The present study shows, for the first time, the structure of the entire eukaryotic apo-Hsp90, along with its intrinsic flexibility. Although large structural rearrangements, leading to partial closure of the Hsp90 dimer, were previously attributed to the binding of nucleotides, our results reveal that they are in fact mainly due to the intrinsic flexibility of Hsp90 dimer. Taking into account the preponderant role of the dynamic nature of the structure of Hsp90, we reconsider the Hsp90 ATPase cycle.

Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.