Cyclic guanosine 3′,5′‐monophosphate (cGMP) is an intracellular signalling molecule involved in many sensory and developmental processes. Synthesis of cGMP from GTP is catalysed 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.
Database
Structural data are available in Protein Data Bank database under the accession number 6SIR.
Enzymes
EC4.6.1.2.
Synthesis of cGMP, an important intracellular signalling molecule, is catalysed by guanylate cyclases. Although detailed structural information is available, current data do not provide a sufficient explanation for the substrate selectivity between guanylate and adenylate cyclases. We present the crystal structure of the GTP‐bound catalytic domain of rhodopsin–GC from Catenaria anguillulae at 1.7 Å resolution. Our study provides insights into potential mechanisms of substrate discrimination and activity regulation.
•Crystal Structure of Adenylosuccinate Lyase Apo form and in complex with AMP from Schistosoma mansoni..•Analysis of the dimer interface and structural stability as a potential chemotherapeutic ...target.•Characterization of SmADSL enzymatic kinetic by isothermal titration calorimetry.
Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5′-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites are formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway.
Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine ...monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s(-1) and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5'-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs.
Cyclic guanosine 3′,5′‐monophosphate (
cGMP
) is an intracellular signalling molecule involved in many sensory and developmental processes. Synthesis of
cGMP
from
GTP
is catalysed 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 (
NC
s) 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
(Rh
GC
) 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.
Database
Structural data are available in Protein Data Bank database under the accession number
6SIR
.
Enzymes
EC4.6.1.2
.
The CD2-CD58 recognition system promotes adhesion and signaling and counters exhaustion in human T cells. We found that CD2 localized to the outer edge of the mature immunological synapse, with ...cellular or artificial APC, in a pattern we refer to as a 'CD2 corolla'. The corolla captured engaged CD28, ICOS, CD226 and SLAM-F1 co-stimulators. The corolla amplified active phosphorylated Src-family kinases (pSFK), LAT and PLC-γ over T cell receptor (TCR) alone. CD2-CD58 interactions in the corolla boosted signaling by 77% as compared with central CD2-CD58 interactions. Engaged PD-1 invaded the CD2 corolla and buffered CD2-mediated amplification of TCR signaling. CD2 numbers and motifs in its cytoplasmic tail controlled corolla formation. CD8
tumor-infiltrating lymphocytes displayed low expression of CD2 in the majority of people with colorectal, endometrial or ovarian cancer. CD2 downregulation may attenuate antitumor T cell responses, with implications for checkpoint immunotherapies.
Production of high quality protein is an essential step for both structural and functional studies. Throughput has increased in the past decade by the use of streamlined workflows with standard ...operating procedures and automation. In this chapter, we describe the Oxford Protein Production Facility (OPPF) pipeline for protein production, from conception, through vector construction, to expression and purification. Results from projects run in the OPPF demonstrate the value of using parallel expression screening of intracellular proteins in both E. coli and insect cells. Transient expression in Human Embryonic Kidney (HEK) cells is used exclusively for production of secreted glycoproteins. Protein purification and quality assessment are independent of the expression system and enable sample preparation to be simplified and streamlined.
•A crystal structure of Schistosoma mansoni HGPRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ is described.•RNA-Seq and WISH data, suggests that some HGPRT isoforms might be involved in ...sexual maturation and reproduction in worms.•S. mansoni has four HGPRT isoforms, here we described the kinetics assays for three of them.
Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the “de novo” purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.
Summary
Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ‐phosphate of ATP to 2′‐deoxythymidine (dThd) forming thymidine monophosphate ...(dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C‐terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine‐derived nucleotides. In addition, we report the X‐ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.
In this study, we establish that the depletion of Trypanosoma brucei thymidine kinase (TbTK) leads to strongly diminished dTTP pools, DNA damage and parasite death. We show that TbTK expression is cell cycle regulated and essential for cell cycle progression. In addition, we report the X‐ray structure of the catalytically active domain of TbTK and identify structural peculiarities within the active site that could be exploited in the process of rational drug design.
Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine ...monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s-1 and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5'-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs.
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