Structural studies of complexes of kallikrein 4 with wild-type and mutated forms of the Kunitz-type inhibitor BbKI

2021 ◽  
Vol 77 (8) ◽  
Author(s):  
Mi Li ◽  
Jaroslav Srp ◽  
Michael Mareš ◽  
Alexander Wlodawer ◽  
Alla Gustchina
Keyword(s):  
Serine Proteases ◽  
Structural Studies ◽  
Wild Type ◽  
Inhibitory Potency ◽  
Crystal Forms ◽  
Space Groups ◽  
Bovine Trypsin ◽  
Kallikrein 4 ◽  
Kunitz Type ◽  
Resolution Structure

Structures of BbKI, a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides, complexed with human kallikrein 4 (KLK4) were determined at medium-to-high resolution in four crystal forms (space groups P3121, P6522, P21 and P61). Although the fold of the protein was virtually identical in all of the crystals, some significant differences were observed in the conformation of Arg64 of BbKI, the residue that occupies the S1 pocket in KLK4. Whereas this residue exhibited two orientations in the highest resolution structure (P3121), making either a canonical trypsin-like interaction with Asp189 of KLK4 or an alternate interaction, only a single alternate orientation was observed in the other three structures. A neighboring disulfide, Cys191–Cys220, was partially or fully broken in all KLK4 structures. Four variants of BbKI in which Arg64 was replaced by Met, Phe, Ala and Asp were expressed and crystallized, and their structures were determined in complex with KLK4. Structures of the Phe and Met variants complexed with bovine trypsin and of the Phe variant complexed with α-chymotrypsin were also determined. Although the inhibitory potency of these variant forms of BbKI was lowered by up to four orders of magnitude, only small changes were seen in the vicinity of the mutated residues. Therefore, a totality of subtle differences in KLK4–BbKI interactions within the fully extended interface in the structures of these variants might be responsible for the observed effect. Screening of the BbKI variants against a panel of serine proteases revealed an altered pattern of inhibitory specificity, which was shifted towards that of chymotrypsin-like proteases for the hydrophobic Phe and Met P1 substitutions. This work reports the first structures of plant Kunitz inhibitors with S1-family serine proteases other than trypsin, as well as new insights into the specificity of inhibition of medically relevant kallikreins.

scholarly journals Structural and Drug Targeting Insights on Mutant p53

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3344
Author(s):  
Ana Sara Gomes ◽  
Helena Ramos ◽  
Alberto Inga ◽  
Emília Sousa ◽  
Lucília Saraiva
Keyword(s):  
Small Molecules ◽  
Drug Targeting ◽  
Normal Function ◽  
Structure Stability ◽  
Mutant P53 ◽  
Structural Studies ◽  
Wild Type ◽  
Tumor Onset ◽  
Long Time ◽  
Stability Dynamics

p53 is a transcription factor with a pivotal role in cell homeostasis and fate. Its impairment is a major event in tumor onset and development. In fact, about half of human cancers bear TP53 mutations that not only halt the normal function of p53, but also may acquire oncogenic gain of functions that favor tumorigenesis. Although considered undruggable for a long time, evidence has proven the capability of many compounds to restore a wild-type (wt)-like function to mutant p53 (mutp53). However, they have not reached the clinic to date. Structural studies have strongly contributed to the knowledge about p53 structure, stability, dynamics, function, and regulation. Importantly, they have afforded relevant insights into wt and mutp53 pharmacology at molecular levels, fostering the design and development of p53-targeted anticancer therapies. Herein, we provide an integrated view of mutp53 regulation, particularly focusing on mutp53 structural traits and on targeting agents capable of its reactivation, including their biological, biochemical and biophysical features. With this, we expect to pave the way for the development of improved small molecules that may advance precision cancer therapy by targeting p53.

scholarly journals CSE4 Genetically Interacts With the Saccharomyces cerevisiae Centromere DNA Elements CDE I and CDE II but Not CDE III: Implications for the Path of the Centromere DNA Around a Cse4p Variant Nucleosome

Genetics ◽  
2000 ◽  
Vol 156 (3) ◽  
pp. 973-981
Author(s):  
Kevin C Keith ◽  
Molly Fitzgerald-Hayes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Loss ◽  
Structural Studies ◽  
Wild Type ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
Histone H3 Variant ◽  
Dna Elements ◽  
Mutant Cells ◽  
Loss Rates

Abstract Each Saccharomyces cerevisiae chromosome contains a single centromere composed of three conserved DNA elements, CDE I, II, and III. The histone H3 variant, Cse4p, is an essential component of the S. cerevisiae centromere and is thought to replace H3 in specialized nucleosomes at the yeast centromere. To investigate the genetic interactions between Cse4p and centromere DNA, we measured the chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA (cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants but expressing wild-type Cse4p, we found that mutations throughout the Cse4p histone-fold domain caused surprisingly large increases in the loss of chromosomes carrying CDE I or CDE II mutant centromeres, but had no effect on chromosomes with CDE III mutant centromeres. Our genetic evidence is consistent with direct interactions between Cse4p and the CDE I-CDE II region of the centromere DNA. On the basis of these and other results from genetic, biochemical, and structural studies, we propose a model that best describes the path of the centromere DNA around a specialized Cse4p-nucleosome.

scholarly journals Ensovibep, a novel trispecific DARPin candidate that protects against SARS-CoV-2 variants

2021 ◽  
Author(s):  
Michael Stumpp
Keyword(s):  
Single Agent ◽  
Standard Of Care ◽  
Cooperative Binding ◽  
Wild Type ◽  
Inhibitory Potency ◽  
Alpha Variant ◽  
The Individual ◽  
Very High ◽  
Comprehensive Study

Abstract SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron, as of December 2021. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19.

scholarly journals Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron

1990 ◽  
Vol 272 (3) ◽  
pp. 805-811 ◽  
Author(s):  
K Ramotar ◽  
B Boyd ◽  
G Tyrrell ◽  
J Gariepy ◽  
C Lingwood ◽  
...  
Keyword(s):  
Culture Medium ◽  
Polymyxin B ◽  
Exchange Chromatography ◽  
Structural Studies ◽  
Wild Type ◽  
Binding Studies ◽  
B Subunit ◽  
Tac Promoter ◽  
Glycolipid Receptor

The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.

Atomic resolution structure of a chimeric DNA–RNA Z-type duplex in complex with Ba2+ions: a case of complicated multi-domain twinning

2016 ◽  
Vol 72 (2) ◽  
pp. 211-223 ◽  
Author(s):  
Miroslaw Gilski ◽  
Pawel Drozdzal ◽  
Ryszard Kierzek ◽  
Mariusz Jaskolski
Keyword(s):  
Molecular Replacement ◽  
Higher Symmetry ◽  
Barium Ions ◽  
Space Groups ◽  
Left Handed ◽  
Crystal Twinning ◽  
Using Data ◽  
High Degree ◽  
Packing Analysis ◽  
Resolution Structure

The self-complementary dCrGdCrGdCrG hexanucleotide, in which not only the pyrimidine/purine bases but also the ribo/deoxy sugars alternate along the sequence, was crystallized in the presence of barium cations in the form of a left-handed Z-type duplex. The asymmetric unit of theP21crystal with a pseudohexagonal lattice contains four chimeric duplexes and 16 partial Ba2+sites. The chimeric (DNA–RNA)2duplexes have novel patterns of hydration and exhibit a high degree of discrete conformational disorder of their sugar-phosphate backbones, which can at least partly be correlated with the fractional occupancies of the barium ions. The crystals of the DNA–RNA chimeric duplex in complex with Ba2+ions and also with Sr2+ions exhibit complicated twinning, which in combination with structural pseudosymmetry made structure determination difficult. The structure could be successfully solved by molecular replacement in space groupsP1 andP21but not in orthorhombic or higher symmetry and, after scrupulous twinning and packing analysis, was refined in space groupP21to anRandRfreeof 11.36 and 16.91%, respectively, using data extending to 1.09 Å resolution. With the crystal structure having monoclinic symmetry, the sixfold crystal twinning is a combination of threefold and twofold rotations. The paper describes the practical aspects of dealing with cases of complicated twinning and pseudosymmetry, and compares the available software tools for the refinement and analysis of such cases.

scholarly journals Mast cells limit extracellular levels of IL-13 via a serglycin proteoglycan-serine protease axis

2012 ◽  
Vol 393 (12) ◽  
pp. 1555-1567 ◽  
Author(s):  
Ida Waern ◽  
Iulia Karlsson ◽  
Michael Thorpe ◽  
Susan M. Schlenner ◽  
Thorsten B. Feyerabend ◽  
...  
Keyword(s):  
Mast Cell ◽  
Serine Protease ◽  
Serine Proteases ◽  
Allergic Inflammation ◽  
Serine Protease Inhibitor ◽  
Local Inflammation ◽  
Wild Type ◽  
Carboxypeptidase A ◽  
Protective Function ◽  
Inactivating Mutation

Abstract Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin, a proteoglycan with heparin side chains. Hence, serglycin-protease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation, whereas serglycin–/– MCs completely lacked this ability. Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist, which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex. Moreover, IL-13 degradation was abrogated in MC-CPA–/– MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein. Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation.

scholarly journals Crystal structures of two furazidin polymorphs revealed by a joint effort of crystal structure prediction and NMR crystallography

2020 ◽  
Vol 76 (3) ◽  
pp. 322-335 ◽  
Author(s):  
Marta K. Dudek ◽  
Piotr Paluch ◽  
Edyta Pindelska
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Structure Determination ◽  
Structure Prediction ◽  
Structural Features ◽  
Crystal Structure Determination ◽  
Crystal Structure Prediction ◽  
Crystal Forms ◽  
Space Groups ◽  
Nmr Crystallography

This work presents the crystal structure determination of two elusive polymorphs of furazidin, an antibacterial agent, employing a combination of crystal structure prediction (CSP) calculations and an NMR crystallography approach. Two previously uncharacterized neat crystal forms, one of which has two symmetry-independent molecules (form I), whereas the other one is a Z′ = 1 polymorph (form II), crystallize in P21/c and P 1 space groups, respectively, and both are built by different conformers, displaying different intermolecular interactions. It is demonstrated that the usage of either CSP or NMR crystallography alone is insufficient to successfully elucidate the above-mentioned crystal structures, especially in the case of the Z′ = 2 polymorph. In addition, cases of serendipitous agreement in terms of 1H or 13C NMR data obtained for the CSP-generated crystal structures different from the ones observed in the laboratory (false-positive matches) are analyzed and described. While for the majority of analyzed crystal structures the obtained agreement with the NMR experiment is indicative of some structural features in common with the experimental structure, the mentioned serendipity observed in exceptional cases points to the necessity of caution when using an NMR crystallography approach in crystal structure determination.

New Crystal Forms and Low Resolution Structure Analysis of 20S Proteasomes from Bovine Liver

2000 ◽  
Vol 127 (6) ◽  
pp. 941-943 ◽  
Author(s):  
Y. Tomisugi ◽  
M. Unno ◽  
T. Mizushima ◽  
Y. Morimoto ◽  
N. Tarmhashi ◽  
...  
Keyword(s):  
Structure Analysis ◽  
Bovine Liver ◽  
Low Resolution ◽  
Crystal Forms ◽  
Resolution Structure

scholarly journals Crystallization of an atypical short-chain dehydrogenase fromVibrio vulnificuslacking the conserved catalytic tetrad

2012 ◽  
Vol 68 (7) ◽  
pp. 771-774 ◽  
Author(s):  
Geraldine Buysschaert ◽  
Kenneth Verstraete ◽  
Savvas N. Savvides ◽  
Bjorn Vergauwen
Keyword(s):  
Protein Sequence ◽  
Scientific Literature ◽  
Vibrio Vulnificus ◽  
Short Chain ◽  
Reduced Form ◽  
Molecular Replacement ◽  
Structural Studies ◽  
Sequence Alignments ◽  
Crystal Forms ◽  
Short Chain Dehydrogenase

Short-chain dehydrogenases/reductases (SDRs) are a rapidly expanding superfamily of enzymes that are found in all kingdoms of life. Hallmarked by a highly conserved Asn-Ser-Tyr-Lys catalytic tetrad, SDRs have a broad substrate spectrum and play diverse roles in key metabolic processes. Locus tag VVA1599 inVibrio vulnificusencodes a short-chain dehydrogenase (hereafter referred to as SDRvv) which lacks the signature catalytic tetrad of SDR members. Structure-based protein sequence alignments have suggested that SDRvv may harbour a unique binding site for its nicotinamide cofactor. To date, structural studies of SDRs with altered catalytic centres are underrepresented in the scientific literature, thus limiting understanding of their spectrum of substrate and cofactor preferences. Here, the expression, purification and crystallization of recombinant SDRvv are presented. Two well diffracting crystal forms could be obtained by cocrystallization in the presence of the reduced form of the phosphorylated nicotinamide cofactor NADPH. The collected data were of sufficient quality for successful structure determination by molecular replacement and subsequent refinement. This work sets the stage for deriving the identity of the natural substrate of SDRvv and the structure–function landscape of typical and atypical SDRs.

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