Does Acinetobacter calcoaceticus glucose dehydrogenase produce self-damaging H2O2?

The soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus has been widely studied and is used, in biosensors, to detect the presence of glucose, taking advantage of its high turnover and insensitivity to molecular oxygen. This approach, however, presents two drawbacks: the enzyme has broad substrate specificity (leading to imprecise blood glucose measurements) and shows instability over time (inferior to other oxidizing glucose enzymes). We report the characterization of two sGDH mutants: the single mutant Y343F and the double mutant D143E/Y343F. The mutants present enzyme selectivity and specificity of 1.2 (Y343F) and 5.7 (D143E/Y343F) times higher for glucose compared with that of the wild-type. Crystallographic experiments, designed to characterize these mutants, surprisingly revealed that the prosthetic group PQQ (pyrroloquinoline quinone), essential for the enzymatic activity, is in a cleaved form for both wild-type and mutant structures. We provide evidence suggesting that the sGDH produces H2O2, the level of production depending on the mutation. In addition, spectroscopic experiments allowed us to follow the self-degradation of the prosthetic group and the disappearance of sGDH's glucose oxidation activity. These studies suggest that the enzyme is sensitive to its self-production of H2O2. We show that the premature aging of sGDH can be slowed down by adding catalase to consume the H2O2 produced, allowing the design of a more stable biosensor over time. Our research opens questions about the mechanism of H2O2 production and the physiological role of this activity by sGDH.

Supramolecular Synthons in Protein-Ligand Frameworks.

Supramolecular synthons, defined as reproducible intermolecular structural units, have greatly aided small molecule crystal engineering. In this paper, we propose that supramolecular synthons guide ligand-mediated protein crystallization. The protein RSL and the macrocycle sulfonato-calix[8]arene cocrystallize in at least four ways. One of these cocrystals is a highly porous cube comprising protein nodes connected by calixarene dimers. We show that mutating an aspartic acid to an asparagine results in two new cubic assemblies that depend also on the crystallization method. One of the new cubic arrangements is mediated by calixarene trimers and has a ∼30% increased cell volume relative to the original crystal with calixarene dimers. Crystals of the sulfonato-calix[8]arene sodium salt were obtained from buffered conditions similar to those used to grow the protein-calix[8]arene cocrystals. X-ray analysis reveals a coordination polymer of the anionic calix[8]arene and sodium cation in which the macrocycle is arranged as staggered stacks of the pleated loop conformation. Remarkably, the calixarene packing arrangement is the same in the simple salt as in the protein cocrystal. With the pleated loop conformation, the calixarene presents an extended surface for binding other calixarenes (oligomerization) as well as binding to a protein patch (biomolecular complexation). Small-angle X-ray scattering data suggest pH-dependent calixarene assembly in solution. Therefore, the calix[8]arene-calix[8]arene structural unit may be regarded as a supramolecular synthon that directs at least two types of protein assembly, suggesting applications in protein crystal engineering.

Disordered regions and folded modules in CAF-1 promote histone deposition in Schizosaccharomyces pombe.

Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the Saccharomyces cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.

Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme.

D-Amino acid residues, found in countless peptides and natural products including ribosomally synthesized and post-translationally modified peptides (RiPPs), are critical for the bioactivity of several antibiotics and toxins. Recently, radical S-adenosyl-L-methionine (SAM) enzymes have emerged as the only biocatalysts capable of installing direct and irreversible epimerization in RiPPs. However, the mechanism underpinning this biochemical process is ill-understood and the structural basis for this post-translational modification remains unknown. Here we report an atomic-resolution crystal structure of a RiPP-modifying radical SAM enzyme in complex with its substrate properly positioned in the active site. Crystallographic snapshots, size-exclusion chromatography-small-angle x-ray scattering, electron paramagnetic resonance spectroscopy and biochemical analyses reveal how epimerizations are installed in RiPPs and support an unprecedented enzyme mechanism for peptide epimerization. Collectively, our study brings unique perspectives on how radical SAM enzymes interact with RiPPs and catalyze post-translational modifications in natural products.

Phosphorylation motif dictates GPCR C-terminal domain conformation and arrestin interaction.

Arrestin-dependent G protein-coupled receptor (GPCR) signaling pathway is regulated by the phosphorylation state of GPCR's C-terminal domain, but the molecular bases of arrestin:receptor interaction are to be further illuminated. Here we investigated the impact of phosphorylation on the conformational features of the C-terminal region from three rhodopsin-like GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR), and the ß2-adernergic receptor (ß2AR). Using phosphomimetic variants, we identified pre-formed secondary structure elements, or short linear motifs (SLiMs), that undergo specific conformational transitions upon phosphorylation. Of importance, such conformational transitions appear to favor arrestin-2 binding. Hence, our results suggest a model in which the phosphorylation-dependent structuration of the GPCR C-terminal regions would modulate arrestin binding and therefore signaling outcomes in arrestin-dependent pathways.

Exploring and strengthening the potential of R-phycocyanin from Nori flakes as a food colourant.

This study aimed to purify, characterise and stabilise the natural food colourant, R-phycocyanin (R-PC), from the red algae Porphyra spp. (Nori). We purified R-PC from dried Nori flakes with a high purity ratio (A618/A280 ≥ 3.4) in native form (α-helix content 53%). SAXS measurements revealed that R-PC is trimeric ((αß)3) in solution. The thermal denaturation of α-helix revealed one transition (Tm at 52 °C), while the pH stability study showed R-PC is stable in the pH range 4-8. The thermal treatment of R-PC at 60 °C has detrimental and irreversible effects on R-PC colour and antioxidant capacity (22 % of residual capacity). However, immobilisation of R-PC within calcium alginate beads completely preserves R-PC colour and mainly retains its antioxidant ability (78 % of residual capacity). Results give new insights into the stability of R-PC and preservation of its purple colour and bioactivity by encapsulation in calcium alginate beads.

Structural transitions in TCTP tumor protein upon binding to the anti-apoptotic protein family member Mcl-1.

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a ß-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.

The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity.

Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases.

Molecular interactions of PCSK9 with an inhibitory nanobody, CAP1 and HLA-C: Functional regulation of LDLR levels.

OBJECTIVE: The liver-derived circulating PCSK9 enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes. PCSK9 inhibition or silencing is presently used in clinics worldwide to reduce LDL-cholesterol, resulting in lower incidence of cardiovascular disease and possibly cancer/metastasis. The mechanism by which the PCSK9-LDLR complex is sorted to degradation compartments is not fully understood. We previously suggested that out of the three M1, M2 and M3 subdomains of the C-terminal Cys/His-rich-domain (CHRD) of PCSK9, only M2 is critical for the activity of extracellular of PCSK9 on cell surface LDLR. This likely implicates the binding of M2 to an unknown membrane-associated "protein X" that would escort the complex to endosomes/lysosomes for degradation. We reported that a nanobody P1.40 binds the M1 and M3 domains of the CHRD and inhibits the function of PCSK9. It was also reported that the cytosolic adenylyl cyclase-associated protein 1 (CAP1) could bind M1 and M3 subdomains and enhance the activity of PCSK9. In this study, we determined the 3-dimensional structure of the CHRD-P1.40 complex to understand the intricate interplay between P1.40, CAP1 and PCSK9 and how they regulate LDLR degradation. METHODS: X-ray diffraction of the CHRD-P1.40 complex was analyzed with a 2.2 Å resolution. The affinity and interaction of PCSK9 or CHRD with P1.40 or CAP1 was analyzed by atomic modeling, site-directed mutagenesis, bio-layer interferometry, expression in hepatic cell lines and immunocytochemistry to monitor LDLR degradation. The CHRD-P1.40 interaction was further analyzed by deep mutational scanning and binding assays to validate the role of predicted critical residues. Conformational changes and atomic models were obtained by small angle X-ray scattering (SAXS). RESULTS: We demonstrate that PCSK9 exists in a closed or open conformation and that P1.40 favors the latter by binding key residues in the M1 and M3 subdomains of the CHRD. Our data show that CAP1 is well secreted by hepatic cells and binds extracellular PCSK9 at distinct residues in the M1 and M3 modules and in the acidic prodomain. CAP1 stabilizes the closed conformation of PCSK9 and prevents P1.40 binding. However, CAP1 siRNA only partially inhibited PCSK9 activity on the LDLR. By modeling the previously reported interaction between M2 and an R-X-E motif in HLA-C, we identified Glu567 and Arg549 as critical M2 residues binding HLA-C. Amazingly, these two residues are also required for the PCSK9-induced LDLR degradation. CONCLUSIONS: The present study reveals that CAP1 enhances the function of PCSK9, likely by twisting the protein into a closed configuration that exposes the M2 subdomain needed for targeting the PCSK9-LDLR complex to degradation compartments. We hypothesize that "protein X", which is expected to guide the LDLR-PCSK9-CAP1 complex to these compartments after endocytosis into clathrin-coated vesicles, is HLA-C or a similar MHC-I family member. This conclusion is supported by the PCSK9 natural loss-of-function Q554E and gain-of-function H553R M2 variants, whose consequences are anticipated by our modeling.

Structure of SpoT reveals evolutionary tuning of catalysis via conformational constraint.

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyzes (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intramolecular regulation of 'long'-stringent factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialization of long RelA-SpoT homologs toward either synthesis or hydrolysis: the short and structured Core of SpoT stabilizes the τ-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the τ-state priming the monofunctional RelA for efficient (p)ppGpp synthesis.

A round-robin approach provides a detailed assessment of biomolecular small-angle scattering data reproducibility and yields consensus curves for benchmarking.

Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1 Å-1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å-1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å-1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.

SEC-SAXS: Experimental set-up and software developments build up a powerful tool.

A monodispersed and ideal solution is a key requirement of BioSAXS to allow one to extract structural information from the recorded pattern. On-line size exclusion chromatography (SEC) marked a major breakthrough, separating particles present in solution according to their size. Scattering curves with identical shape under an elution peak can be averaged and further processed free from contamination. However, this is not always straightforward, separation is often incomplete. Software have been developed to deconvolve the contributions from the different species (molecules or oligomeric forms) within the sample. In this chapter, we present the general workflow of a SEC-SAXS experiment. We present recent instrumental and data analysis developments that have improved the quality of recorded data, extended the potential of SEC-SAXS and turned it into a mainstream approach. We report a comparative analysis of two macromolecular systems using various deconvolution approaches that have been developed over the last years. Parallel analysis appears to be the best cross-validation method to assess the reliability of the reconstructed isolated species patterns that can safely be used as a support for meaningful molecular modeling.

Medical contrast agents as promising tools for biomacromolecular SAXS experiments.

Small-angle X-ray scattering (SAXS) has become an indispensable tool in structural biology, complementing atomic-resolution techniques. It is sensitive to the electron-density difference between solubilized biomacromolecules and the buffer, and provides information on molecular masses, particle dimensions and interactions, low-resolution conformations and pair distance-distribution functions. When SAXS data are recorded at multiple contrasts, i.e. at different solvent electron densities, it is possible to probe, in addition to their overall shape, the internal electron-density profile of biomacromolecular assemblies. Unfortunately, contrast-variation SAXS has been limited by the range of solvent electron densities attainable using conventional co-solutes (for example sugars, glycerol and salt) and by the fact that some biological systems are destabilized in their presence. Here, SAXS contrast data from an oligomeric protein and a protein-RNA complex are presented in the presence of iohexol and Gd-HPDO3A, two electron-rich molecules that are used in biomedical imaging and that belong to the families of iodinated and lanthanide-based complexes, respectively. Moderate concentrations of both molecules allowed solvent electron densities matching those of proteins to be attained. While iohexol yielded higher solvent electron densities (per mole), it interacted specifically with the oligomeric protein and precipitated the protein-RNA complex. Gd-HPDO3A, while less efficient (per mole), did not disrupt the structural integrity of either system, and atomic models could be compared with the SAXS data. Due to their elevated solubility and electron density, their chemical inertness, as well as the possibility of altering their physico-chemical properties, lanthanide-based complexes represent a class of molecules with promising potential for contrast-variation SAXS experiments on diverse biomacromolecular systems.

Oligomerization processes limit photoactivation and recovery of the orange carotenoid protein.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection by quenching of the excess of light-harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy-quenching activity. Here, we probed photoinduced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation a numbed state forms, which remains in a non-photoexcitable structural state for at least ≈0.5 µs after absorption of a first photon.

Mapping the deformability of natural and designed cellulosomes in solution.

BACKGROUND: Natural cellulosome multi-enzyme complexes, their components, and engineered 'designer cellulosomes' (DCs) promise an efficient means of breaking down cellulosic substrates into valuable biofuel products. Their broad uptake in biotechnology relies on boosting proximity-based synergy among the resident enzymes, but the modular architecture challenges structure determination and rational design. RESULTS: We used small angle X-ray scattering combined with molecular modeling to study the solution structure of cellulosomal components. These include three dockerin-bearing cellulases with distinct substrate specificities, original scaffoldins from the human gut bacterium Ruminococcus champanellensis (ScaA, ScaH and ScaK) and a trivalent cohesin-bearing designer scaffoldin (Scaf20L), followed by cellulosomal complexes comprising these components, and the nonavalent fully loaded Clostridium thermocellum CipA in complex with Cel8A from the same bacterium. The size analysis of Rg and Dmax values deduced from the scattering curves and corresponding molecular models highlight their variable aspects, depending on composition, size and spatial organization of the objects in solution. CONCLUSIONS: Our data quantifies variability of form and compactness of cellulosomal components in solution and confirms that this native plasticity may well be related to speciation with respect to the substrate that is targeted. By showing that scaffoldins or components display enhanced compactness compared to the free objects, we provide new routes to rationally enhance their stability and performance in their environment of action.

Structural Insights into the Intrinsically Disordered GPCR C-Terminal Region, Major Actor in Arrestin-GPCR Interaction.

Arrestin-dependent pathways are a central component of G protein-coupled receptor (GPCRs) signaling. However, the molecular processes regulating arrestin binding are to be further illuminated, in particular with regard to the structural impact of GPCR C-terminal disordered regions. Here, we used an integrated biophysical strategy to describe the basal conformations of the C-terminal domains of three class A GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR) and the ß2-adernergic receptor (ß2AR). By doing so, we revealed the presence of transient secondary structures in these regions that are potentially involved in the interaction with arrestin. These secondary structure elements differ from those described in the literature in interaction with arrestin. This suggests a mechanism where the secondary structure conformational preferences in the C-terminal regions of GPCRs could be a central feature for optimizing arrestins recognition.

A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study.

Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in drosophila. Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, we studied full-length SuFu's structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.

Revisiting the Molecular Interactions between the Tumor Protein TCTP and the Drugs Sertraline/Thioridazine.

TCTP protein is a pharmacological target in cancer and TCTP inhibitors such as sertraline have been evaluated in clinical trials. The direct interaction of TCTP with the drugs sertraline and thioridazine has been reported in vitro by SPR experiments to be in the ∼30-50 µM Kd range (Amson et al. Nature Med 2012), supporting a TCTP-dependent mode of action of the drugs on tumor cells. However, the molecular details of the interaction remain elusive although they are crucial to improve the efforts of on-going medicinal chemistry. In addition, TCTP can be phosphorylated by the Plk-1 kinase, which is indicative of poor prognosis in several cancers. The impact of phosphorylation on TCTP structure/dynamics and binding with therapeutical ligands remains unexplored. Here, we combined NMR, TSA, SPR, BLI and ITC techniques to probe the molecular interactions between TCTP with the drugs sertraline and thioridazine. We reveal that drug binding is much weaker than reported with an apparent ∼mM Kd and leads to protein destabilization that obscured the analysis of the published SPR data. We further demonstrate by NMR and SAXS that TCTP S46 phosphorylation does not promote tighter interaction between TCTP and sertraline. Accordingly, we question the supported model in which sertraline and thioridazine directly interact with isolated TCTP in tumor cells and discuss alternative modes of action for the drugs in light of current literature.

Porphyrin Binding and Irradiation Promote G-Quadruplex DNA Dimeric Structure.

Nucleic acid sequences rich in guanines can organize into noncanonical DNA G-quadruplexes (G4s) of variable size. The design of small molecules stabilizing the structure of G4s is a rapidly growing area for the development of novel anticancer therapeutic strategies and bottom-up nanotechnologies. Among a multitude of binders, porphyrins are very attractive due to their light activation that can make them valuable conformational regulators of G4s. Here, a structure-based strategy, integrating complementary probes, is employed to study the interaction between TMPyP4 porphyrin and a 22-base human telomeric sequence (Tel22) before and after irradiation with blue light. Porphyrin binding is discovered to promote Tel22 dimerization, while light irradiation of the Tel22-TMPyP4 complex controls dimer fraction. Such a change in quaternary structure is found to be strictly correlated with modifications at the secondary structure level, thus providing an unprecedented link between the degree of dimerization and the underlying conformational changes in G4s.

Exploring molecular determinants of polysaccharide lyase family 6-1 enzyme activity.

The polysaccharide lyase family 6 (PL6) represents one of the 41 polysaccharide lyase families classified in the CAZy database with the vast majority of its members being alginate lyases grouped into three subfamilies, PL6_1-3. To decipher the mode of recognition and action of the enzymes belonging to subfamily PL6_1, we solved the crystal structures of Pedsa0632, Patl3640, Pedsa3628 and Pedsa3807, which all show different substrate specificities and mode of action (endo-/exolyase). Thorough exploration of the structures of Pedsa0632 and Patl3640 in complex with their substrates as well as docking experiments confirms that the conserved residues in subsites -1 to +3 of the catalytic site form a common platform that can accommodate various types of alginate in a very similar manner but with a series of original adaptations bringing them their specificities of action. From comparative studies with existing structures of PL6_1 alginate lyases, we observe that in the right-handed parallel ß-helix fold shared by all these enzymes, the substrate-binding site harbors the same overall conserved structures and organization. Despite this apparent similarity, it appears that members of the PL6_1 subfamily specifically accommodate and catalyze the degradation of different alginates suggesting that this common platform is actually a highly adaptable and specific tool.