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When the conventional vapor-diffusion techniques of protein crystallization do not yield crystals of sufficient quality, it does not need to lead to hopelessness as well. Instead, we can apply advanced crystallization techniques such as capillary counter-diffusion and micro-seed matrix screening. We applied these methods to problematic proteins whose crystallization by basic methods was not successful. We selected proteins of our interest, whose revealed structure would be beneficial in addition to their use in validation of the advanced crystallization methods as tools for proteins resisting crystallization by more conventional crystallization methods. The protein targets specifically are: 1. Medicinally relevant human enzymes: purine nucleoside phosphorylases (PNP) in complex with inhibitors and cyclic GMP-AMP synthase (cGAS) 2. A representative of generally difficult-to-crystallize protein-DNA complexes: lactate-utilization repressor (LutR) from Bacillus subtilis in the complex with its DNA operator. Poster will present the results we have achieved in this still-going project. The project was supported by Mobility Plus project number of Czech Academy of Sciences (CSIC-22-02) and the i-LINK 2021 program of the Spanish National Research Council (CSIC) LINKC20027.

The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help to overcome the host's defence during host–parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken in the present study. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and was found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 Å (1 Å=0.1 nm) and was used to describe the structure–inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the pro-inflammatory cytokines tumour necrosis factor α and interleukin-12, and proliferation of antigen-specific CD4+ T-cells. This suggests that OmC2 may suppress the host's adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases

Two previously reported holoprotein crystal forms of the flavodoxin-like E. coli protein WrbA, diffracting to 2.6 and 2.0 Å resolution, and new crystals of WrbA apoprotein diffracting to 1.85 Å, are refined and analysed comparatively through the lens of flavodoxin structures. The results indicate that differences between apo- and holoWrbA crystal structures are manifested on many levels of protein organization as well as in the FMN-binding sites. Evaluation of the influence of crystal contacts by comparison of lattice packing reveals the protein's global response to FMN binding. Structural changes upon cofactor binding are compared with the monomeric flavodoxins. Topologically non-equivalent residues undergo remarkably similar local structural changes upon FMN binding to WrbA or to flavodoxin, despite differences in multimeric organization and residue types at the binding sites. Analysis of the three crystal structures described here, together with flavodoxin structures, rationalizes functional similarities and differences of the WrbAs relative to flavodoxins, leading to a new understanding of the defining features of WrbAs. The results suggest that WrbAs are not a remote and unusual branch of the flavodoxin family as previously thought but rather a central member with unifying structural features

The crystal structure of the flavodoxin-like protein WrbA with oxidized FMN bound reveals a close relationship to mammalian NAD(P)H:quinone oxidoreductase, Nqo1. Structural comparison of WrbA, flavodoxin, and Nqo1 indicates how the twisted open-sheet fold of flavodoxins is elaborated to form multimers that extend catalytic function from one-electron transfer between protein partners using FMN to two-electron reduction of xenobiotics using FAD. The structure suggests a novel physiological role for WrbA and Nqo1.

The flavoprotein WrbA from Escherichia coli is considered to be the prototype of a new family of multimeric flavodoxin-like proteins that are implicated in cell protection against oxidative stress. The present study is aimed at structural characterization of the E. coli protein with respect to its recently revealed oxidoreductase activity. Crystals of WrbA holoprotein in complex with the oxidized flavin cofactor (FMN) were obtained using standard vapour-diffusion techniques. Deep yellow tetragonal crystals obtained from differing crystallization conditions display different space groups and unit-cell parameters. X-ray crystal structures of the WrbA holoprotein have been determined to resolutions of 2.0 and 2.6 angstrom