Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R

In human cells, thymidylate synthase (TS) provides the only source of 2’-deoxythymidyne-5’-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking the hTS active site has limitations due to the onset of resistance in cancer cells, requiring the identification of new strategies to effectively inhibit this enzyme. Human TS works as an obligate homodimer, making the inter-subunit interface an attractive targetable area. Here, we report the design and investigation of a new hTS variant, in which Gln62, located at the dimer interface, has been replaced by arginine in order to destabilize the enzyme quaternary assembly. The hTS Q62R variant has been characterized though kinetic assay, thermal denaturation analysis and X-ray crystallography. Our results provide evidence that hTS Q62R has a reduced melting temperature. The effective destabilization of the TS quaternary structure is also confirmed by structural analysis, showing that the introduced mutation induces a slight aperture of the hTS dimer. The generation of hTS variants having a more accessible interface area can facilitate the screening of interface-targeting molecules, providing key information for the rational design of innovative hTS interface inhibitors.

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Structural and Functional Characterization of the Human Thymidylate Synthase (hTS) Interface Variant R175C, New Perspectives for the Development of hTS Inhibitors

Molecules ◽

10.3390/molecules24071362 ◽

2019 ◽

Vol 24 (7) ◽

pp. 1362

Author(s):

Cecilia Pozzi ◽

Stefania Ferrari ◽

Rosaria Luciani ◽

Maria Costi ◽

Stefano Mangani

Keyword(s):

Thymidylate Synthase ◽

Active Site ◽

Functional Characterization ◽

Binding Mode ◽

Anticancer Chemotherapy ◽

Innovative Strategies ◽

X Ray Crystallography ◽

Site Targeting ◽

New Strategies

Human thymidylate synthase (hTS) is pivotal for cell survival and proliferation, indeed it provides the only synthetic source of dTMP, required for DNA biosynthesis. hTS represents a validated target for anticancer chemotherapy. However, active site-targeting drugs towards hTS have limitations connected to the onset of resistance. Thus, new strategies have to be applied to effectively target hTS without inducing resistance in cancer cells. Here, we report the generation and the functional and structural characterization of a new hTS interface variant in which Arg175 is replaced by a cysteine. Arg175 is located at the interface of the hTS obligate homodimer and protrudes inside the active site of the partner subunit, in which it provides a fundamental contribution for substrate binding. Indeed, the R175C variant results catalytically inactive. The introduction of a cysteine at the dimer interface is functional for development of new hTS inhibitors through innovative strategies, such as the tethering approach. Structural analysis, performed through X-ray crystallography, has revealed that a cofactor derivative is entrapped inside the catalytic cavity of the hTS R175C variant. The peculiar binding mode of the cofactor analogue suggests new clues exploitable for the design of new hTS inhibitors.

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Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: Role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase

Proteins Structure Function and Bioinformatics ◽

10.1002/(sici)1097-0134(19990215)34:3<356::aid-prot8>3.0.co;2-o ◽

1999 ◽

Vol 34 (3) ◽

pp. 356-368 ◽

Cited By ~ 7

Author(s):

Variath Prasanna ◽

B. Gopal ◽

M.R.N. Murthy ◽

Daniel V. Santi ◽

Padmanabhan Balaram

Keyword(s):

Amino Acid ◽

Thymidylate Synthase ◽

Amino Acid Substitutions ◽

Dimer Interface ◽

Subunit Interface ◽

Dimeric Protein ◽

The Stability

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Targeting HIV using Structure-Based Rational Design of Antibodies

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314098805 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C119-C119

Author(s):

Ron Diskin ◽

Johannes Scheid ◽

Anthony West ◽

Florian Klein ◽

Michel Nussenzweig ◽

...

Keyword(s):

Binding Site ◽

Neutralizing Antibodies ◽

Rational Design ◽

Passive Immunization ◽

Broadly Neutralizing Antibodies ◽

X Ray Crystallography ◽

Cd4 Binding Site ◽

Topical Microbicides ◽

Anti Hiv ◽

New Strategies

Isolating and studying broadly anti HIV neutralizing antibodies from infected individuals is a major effort in the combat against HIV. We hope to gain better understanding for vaccine design by elucidating the exact epitope of such antibodies. Furthermore, by providing evermore potent and broadly neutralizing antibodies new strategies like passive immunization or using antibodies as topical microbicides for HIV prevention become feasible. One class of promising antibodies is anti CD4 binding site antibodies. Such antibodies can show exceptional potency and breadth. We used X-ray crystallography to study the structure of NIH45-46, one of the most potent anti CD4 binding site antibodies ever described in complex with gp120, the HIV receptor binding domain. Our structural analysis identified a region in the inner domain of gp120 that is important for the binding and neutralization of NIH45-46 (Diskin et al., 2011). This region is contacted by a four-residue insertion in CDRH3 that does not exist in VRC01, a less potent clonal member of NIH45-46. We further used structure-based rational design to improve NIH45-46. By replacing Gly54 in the CDRH2 with a tryptophan we allowed NIH45-46G54W to bind a unique hydrophobic pocket on the surface of gp120 that is normally accommodating Phe43 of CD4. Remarkably, NIH45-46G54W shows increase both in breadth and potency compared with NIH45-46 (Diskin et al., 2011). We took this approach further and engineered NIH45-46G54W to bind a conserved glycan on gp120. This effort resulted with the most potent and broadly neutralizing anti HIV antibody ever described (Diskin et al., 2013). I will present this work and additional efforts to modify NIH45-46 to accommodate potential escape mutations of HIV.

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Hierarchy of π-Stacking Determines the Conformational Preference of Bis-Squaraines

10.26434/chemrxiv.13041830.v1 ◽

2020 ◽

Author(s):

Abhishek Singh ◽

Reman K. Singh ◽

G Naresh Patwari

Keyword(s):

Hydrogen Bonding ◽

Rational Design ◽

Biological Molecules ◽

Conformational Space ◽

X Ray Crystallography ◽

Simple Strategy ◽

Induced Folding ◽

Π Stacking ◽

Varying Length ◽

Dynamics Simulations

The rational design of conformationally controlled foldable modules can lead to a deeper insight into the conformational space of complex biological molecules where non-covalent interactions such as hydrogen bonding and π-stacking are known to play a pivotal role. Squaramides are known to have excellent hydrogen bonding capabilities and hence, are ideal molecules for designing foldable modules that can mimic the secondary structures of bio-molecules. The π-stacking induced folding of bis-squaraines tethered using aliphatic primary and secondary-diamine linkers of varying length is explored with a simple strategy of invoking small perturbations involving the length linkers and degree of substitution. Solution phase NMR investigations in combination with molecular dynamics simulations suggest that bis-squaraines predominantly exist as extended conformations. Structures elucidated by X-ray crystallography confirmed a variety of folded and extended secondary conformations including hairpin turns and 𝛽-sheets which are determined by the hierarchy of π-stacking relative to N–H···O hydrogen bonds.

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Hierarchy of π-Stacking Determines the Conformational Preference of Bis-Squaraines

10.26434/chemrxiv.13041830 ◽

2020 ◽

Author(s):

Abhishek Singh ◽

Reman K. Singh ◽

G Naresh Patwari

Keyword(s):

Hydrogen Bonding ◽

Rational Design ◽

Biological Molecules ◽

Conformational Space ◽

X Ray Crystallography ◽

Simple Strategy ◽

Induced Folding ◽

Π Stacking ◽

Varying Length ◽

Dynamics Simulations

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Virtual Screening and X-ray Crystallography Identify Non-Substrate Analog Inhibitors of Flavin-Dependent Thymidylate Synthase

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.6b00977 ◽

2016 ◽

Vol 59 (19) ◽

pp. 9269-9275 ◽

Cited By ~ 12

Author(s):

Rosaria Luciani ◽

Puneet Saxena ◽

Sachin Surade ◽

Matteo Santucci ◽

Alberto Venturelli ◽

...

Keyword(s):

Virtual Screening ◽

Thymidylate Synthase ◽

X Ray ◽

X Ray Crystallography ◽

Substrate Analog

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Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa037 ◽

2021 ◽

Vol 85 (2) ◽

pp. 297-306

Author(s):

Zui Fujimoto ◽

Le Thi Thu Hong ◽

Naomi Kishine ◽

Nobuhiro Suzuki ◽

Keitarou Kimura

Keyword(s):

Bacillus Subtilis ◽

Quaternary Structure ◽

Single Amino Acid ◽

Wild Type ◽

Amino Acid Mutation ◽

X Ray Crystallography ◽

Ligand Binding Sites ◽

Potential Ligand ◽

Single Amino Acid Mutation

ABSTRACT Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth β-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

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Examining the structure of the mature amyloid fibril

Biochemical Society Transactions ◽

10.1042/bst0300521 ◽

2002 ◽

Vol 30 (4) ◽

pp. 521-525 ◽

Cited By ~ 43

Author(s):

O. S. Makin ◽

L. C. Serpell

Keyword(s):

Self Assembly ◽

Rational Design ◽

Amyloid Fibrils ◽

Structural Information ◽

X Ray ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Fibre Diffraction ◽

Complementary Techniques ◽

Mature Fibril

The pathogenesis of the group of diseases known collectively as the amyloidoses is characterized by the deposition of insoluble amyloid fibrils. These are straight, unbranching structures about 70–120 å (1 å = 0.1 nm) in diameter and of indeterminate length formed by the self-assembly of a diverse group of normally soluble proteins. Knowledge of the structure of these fibrils is necessary for the understanding of their abnormal assembly and deposition, possibly leading to the rational design of therapeutic agents for their prevention or disaggregation. Structural elucidation is impeded by fibril insolubility and inability to crystallize, thus preventing the use of X-ray crystallography and solution NMR. CD, Fourier-transform infrared spectroscopy and light scattering have been used in the study of the mechanism of fibril formation. This review concentrates on the structural information about the final, mature fibril and in particular the complementary techniques of cryo-electron microscopy, solid-state NMR and X-ray fibre diffraction.

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Functional CBS modules make IMPDHs octameric

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314087166 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1283-C1283

Author(s):

Gilles Labesse ◽

Thomas Alexandre ◽

Laurène Vaupré ◽

Isabelle Salard-Arnaud ◽

Joséphine Lai Kee Him ◽

...

Keyword(s):

Analytical Ultracentrifugation ◽

Quaternary Structure ◽

Catalytic Domain ◽

Structural Data ◽

Site Directed Mutagenesis ◽

X Ray Crystallography ◽

Binding Pockets ◽

Essential Enzyme ◽

Cbs Domains

Inosine-5'-monophosphate dehydrogenase (1, 2) (IMPDH) is a major target for antiviral, antiparasitic, antileukemic and immunosuppressive therapies. It is an ubiquitous and essential enzyme for the biosynthesis of guanosine nucleotides. Up to now, IMPDHs have been reported as tetrameric enzymes harbouring a catalytic domain and a tandem of cystathionine-ß-synthase (CBS) modules. The latter had no precise function assigned despite their nearly absolute conservation among IMPDHs and consistent indication of their importance in vivo. The aim of our study was to provide evidence for the role of the CBS modules on the quaternary structure and on the regulation of IMPDHs. A multidisciplinary approach involving enzymology, site-directed mutagenesis, analytical ultracentrifugation, X-ray crystallography, SAXS, cryo-electron microscopy and molecular modelling allowed us to demonstrate that the Pseudomonas aeruginosa IMPDH is functionally active as an octamer and allosterically regulated by MgATP via each CBS module. Revisiting deposited structural data, we found this newly discovered octameric organization conserved in other IMPDH structures. Meanwhile, we demonstrated that the human IMPDH1 formed two distinct octamers that can pile up into isolated fibres in the presence of MgATP while its pathogenic mutant D226N, localised into the CBS domains, appeared to form massively aggregating fibres. The dramatic impact of this mutation could explain the severe retinopathy adRP10. Our data (3) revealed for the first time that IMPDH has an octameric architecture modulated by MgATP binding to the CBS modules, inducing large structural rearrangements. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or/and macromolecular assembly. Targeting the conserved effector binding pockets identified within the CBS modules might be promising to develop antibacterial compounds or drugs to counter retinopathy onset.

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