I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[20229]
Open Access
Abstract: The PII‐like protein CutA is annotated as being involved in Cu2+ tolerance, based on analysis of Escherichia coli mutants. However, the precise cellular function of CutA remains unclear. Our bioinformatic analysis reveals that CutA proteins are universally distributed across all domains of life. Based on sequence‐based clustering, we chose representative cyanobacterial CutA proteins for physiological, biochemical, and structural characterization and examined their involvement in heavy metal tolerance, by generating CutA mutants in filamentous Nostoc sp. and in unicellular Synechococcus elongatus . However, we were unable to find any involvement of cyanobacterial CutA in metal tolerance under various conditions. This prompted us to re‐examine experimentally the role of CutA in protecting E. coli from Cu2+. Since we found no effect on copper tolerance, we conclude that CutA plays a different role that is not involved in metal protection. We resolved high‐resolution CutA structures from Nostoc and S. elongatus . Similarly to their counterpart from E. coli and to canonical PII proteins, cyanobacterial CutA proteins are trimeric in solution and in crystal structure; however, no binding affinity for small signaling molecules or for Cu2+ could be detected. The clefts between the CutA subunits, corresponding to the binding pockets of PII proteins, are formed by conserved aromatic and charged residues, suggesting a conserved binding/signaling function for CutA. In fact, we find binding of organic Bis‐Tris/MES molecules in CutA crystal structures, revealing a strong tendency of these pockets to accommodate cargo. This highlights the need to search for the potential physiological ligands and for their signaling functions upon binding to CutA.
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Jul 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15991]
Open Access
Abstract: Cu‐containing nitrite reductases that convert NO2‐ to NO are critical enzymes in nitrogen‐based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4‐domain CuNiR that has both cytochrome and cupredoxin domains fused at the N‐terminus and the other, a classical 2‐domain CuNiR (Br2DNiR). We report the first enzymatic studies of a novel 4‐domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3‐ and 2‐domain variants, and Br2DNiR revealing up to ~500‐fold difference in catalytic efficiency in comparison with classical 2‐domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self‐contained donor‐acceptor system, we demonstrate that 4‐domain BrNiR utilises N‐terminal tethering for down‐regulating enzymatic activity instead. Both Br2DNiR, as well as engineered 2‐domain variant of BrNiR (Δ(Cytc‐Cup) BrNiR), have 3 to 5 % NiR activity compared to the well‐characterized 2‐domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc‐Cup) BrNiR and Br2DNiR with classical 2‐domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally‐occurring 4‐domain CuNiR and its engineered 3‐ and 2‐domain variants.The reverse protein engineering approach utilized here provides has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes.
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Apr 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19190]
Abstract: β‐propeller proteins are common in nature, where they are observed to adopt 4‐ to 10‐fold internal rotational pseudosymmetry. This size diversity can be explained by the evolutionary process of gene duplication and fusion. In this study we investigated a distorted β‐propeller protein, an apparent intermediate between two symmetries. From this template, we created a perfectly symmetric 9‐bladed β‐propeller named Cake, using computational design and ancestral sequence reconstruction. The designed repeat sequence was found to be capable of generating both 8‐fold and 9‐fold propellers which are highly stable. Cake variants with 2 to 10 identical copies of the repeat sequence were characterized by X‐ray crystallography and in solution. They were found to be highly stable, and to self‐assemble into 8‐ or 9‐ fold symmetrical propellers. These findings show that the β‐propeller fold allows sufficient structural plasticity to permit a given blade to assemble different forms, a transition from even to odd changes in blade number, and provide a potential explanation for the wide diversity of repeat numbers observed in natural propeller proteins.
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Apr 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Vibrio spp. play a vital role in the recycling of chitin in oceans, but several Vibrio strains are highly infectious to aquatic animals and humans. These bacteria require chitin for growth; thus, potent inhibitors of chitin‐degrading enzymes could serve as candidate drugs against Vibrio infections. This study examined NAG‐thiazoline (NGT)‐mediated inhibition of a recombinantly expressed GH20 β‐N‐acetylglucosaminidase, namely VhGlcNAcase from Vibrio campbellii (formerly V. harveyi) ATCC BAA‐1116. NGT strongly inhibited VhGlcNAcase with an IC50 of 11.9 ± 1.0 μm and Ki 62 ± 3 µm, respectively. NGT was also found to completely inhibit the growth of V. campbellii strain 650 with an minimal inhibitory concentration value of 0.5 µm. ITC data analysis showed direct binding of NGT to VhGlcNAcase with a Kd of 32 ± 1.2 μm. The observed ΔG°binding of −7.56 kcal·mol−1 is the result of a large negative enthalpy change and a small positive entropic compensation, suggesting that NGT binding is enthalpy‐driven. The structural complex shows that NGT fully occupies the substrate‐binding pocket of VhGlcNAcase and makes an exclusive hydrogen bond network, as well as hydrophobic interactions with the conserved residues around the −1 subsite. Our results strongly suggest that NGT could serve as an excellent scaffold for further development of antimicrobial agents against Vibrio infections.
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Mar 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[1225]
Open Access
Abstract: Trypanosomatids possess glycosome organelles that contain much of the glycolytic machinery, including phosphofructokinase (PFK). We present kinetic and structural data for PFK from three human pathogenic trypanosomatids, illustrating intriguing differences that may reflect evolutionary adaptations to differing ecological niches. The activity of Leishmania PFK – to a much larger extent than Trypanosoma PFK – is reliant on AMP for activity regulation, with 1 mm AMP increasing the L. infantum PFK (LiPFK) kcat/K0.5F6P value by 10‐fold, compared to only a 1.3‐ and 1.4‐fold increase for T. cruzi and T. brucei PFK, respectively. We also show that Leishmania PFK melts at a significantly lower (> 15 °C) temperature than Trypanosoma PFKs and that addition of either AMP or ATP results in a marked stabilization of the protein. Sequence comparisons of Trypanosoma spp. and Leishmania spp. show that divergence of the two genera involved amino acid substitutions that occur in the enzyme’s ‘reaching arms’ and ‘embracing arms’ that determine tetramer stability. The dramatic effects of AMP on Leishmania activity compared with the Trypanosoma PFKs may be explained by differences between the T‐to‐R equilibria for the two families, with the low‐melting Leishmania PFK favouring the flexible inactive T‐state in the absence of AMP. Sequence comparisons along with the enzymatic and structural data presented here also suggest there was a loss of AMP‐dependent regulation in Trypanosoma species rather than gain of this characteristic in Leishmania species and that AMP acts as a key regulator in Leishmania governing the balance between glycolysis and gluconeogenesis.
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Jan 2020
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[16289]
Abstract: The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix that includes the Golgi reassembly and stacking protein (GRASP), which was shown to be involved in cisternae stacking and lateral linkage in metazoan. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different binding partners. The conserved N terminus of the GRASP family includes two PSD‐95, DLG, and ZO‐1 (PDZ) domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content. However, PDZ1 alone mediates nearly all interactions between GRASPs and their partners. In this work, NMR, synchrotron radiation CD, and molecular dynamics (MD) were used to examine the structure, flexibility, and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual β3α1β4β5α2β6β1β2 secondary structural arrangement and NMR data indicate that the PDZ1 binding pocket is formed by a stable β2‐strand and a more flexible and unstable α2‐helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using MD simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner‐dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.
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Jan 2020
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I03-Macromolecular Crystallography
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Abstract: Coproporphyrin ferrochelatases (CpfCs, EC 4.99.1.9) insert ferrous iron into coproporphyrin III yielding coproheme. CpfCs are utilized by prokaryotic, mainly monoderm (Gram‐positive) bacteria within the recently detected coproporphyrin‐dependent heme biosynthesis pathway. Here we present a comprehensive study on CpfC from Listeria monocytogenes (LmCpfC) including the first crystal structure of a coproheme‐bound CpfC. Comparison of crystal structures of apo‐LmCpfC and coproheme‐LmCpfC allowed identification of structural rearrangements and of amino acids involved in tetrapyrrole macrocycle and Fe2+ binding. Differential scanning calorimetry of apo‐, coproporphyrin III‐ and coproheme‐LmCpfC underline the pronounced non‐covalent interaction of both coproporphyrin and coproheme with the protein (ΔTm = 11 °C compared to apo‐LmCpfC) which includes the propionates (p2, p4, p6, p7) and the amino acids Arg29, Arg45, Tyr46, Ser53 and Tyr124. Furthermore, the thermodynamics and kinetics of coproporphyrin III and coproheme binding to apo‐LmCpfC is presented as well as the kinetics of insertion of ferrous iron into coproporphyrin III‐LmCpfC that immediately leads to formation of ferric coproheme‐LmCpfC (kcat/KM = 4.7 × 105 M‐1 s‐1). We compare the crystal structure of coproheme‐LmCpfC with available structures of CpfCs with artificial tetrapyrrole macrocycles and discuss our data on substrate binding, iron insertion and substrate release in the context of the coproporphyrin‐dependent heme biosynthesis pathway.
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Dec 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Diamond Proposal Number(s):
[16818, 19737, 19485]
Open Access
Abstract: Cyclic guanosine 3′,5′‐monophosphate (cGMP) is an intracellular signaling molecule involved in many sensory and developmental processes. Synthesis of cGMP from GTP is catalyzed by guanylate cyclase (GC) in a reaction analogous to cAMP formation by adenylate cyclase (AC). Although detailed structural information is available on the catalytic region of nucleotidyl cyclases (NCs) in various states, these atomic models do not provide a sufficient explanation for the substrate selectivity between GC and AC family members. Detailed structural information on the GC domain in its active conformation is largely missing and no crystal structure of a GTP‐bound wild‐type GC domain has been published to date. Here, we describe the crystal structure of the catalytic domain of rhodopsin‐GC (RhGC) from Catenaria anguillulae in complex with GTP at 1.7 Å resolution. Our study reveals the organization of a eukaryotic GC domain in its active conformation. We observe that the binding mode of the substrate GTP is similar to that of AC–ATP interaction, although surprisingly not all of the interactions predicted to be responsible for base recognition are present. The structure provides insights into potential mechanisms of substrate discrimination and activity regulation that may be common to all class III purine NCs.
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Dec 2019
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I02-Macromolecular Crystallography
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Diana O.
Ribeiro
,
Aldino
Viegas
,
Virgínia M. R.
Pires
,
João
Medeiros‐silva
,
Pedro
Bule
,
Wengang
Chai
,
Filipa
Marcelo
,
Carlos M. G. A.
Fontes
,
Eurico J.
Cabrita
,
Angelina S.
Palma
,
Ana Luisa
Carvalho
Diamond Proposal Number(s):
[16609]
Abstract: Understanding the specific molecular interactions between proteins and β1,3‐1,4‐mixed‐linked d‐glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate‐binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3‐1,4‐mixed‐linked over β1,4‐linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3‐1,4‐linked glucose oligosaccharides showed that both the chain length and the position of the β1,3‐linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site‐directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3‐1,4‐mixed‐linked glucans. This is mediated by a conformation–selection mechanism of the ligand in the binding cleft through CH‐π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3‐linkage at the reducing end and at specific positions along the β1,4‐linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed‐linked β‐glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture.
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Dec 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12788]
Open Access
Abstract: In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three‐step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 → C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an ‘aldol switch’ is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable ‘aldol switch’ consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue‐specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high‐resolution structural analysis of multiple enzyme homologues with classical site‐directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes.
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Oct 2019
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