Kaspars Tars

Latvian Biomedical Research and Study Centre | LBMC · Protein engineering

Lyme disease is the most prevalent vector-borne infectious disease in Europe and the USA. Borrelia burgdorferi , as the causative agent of Lyme disease, is transmitted to the mammalian host during the tick blood meal. To adapt to the different encountered environments, Borrelia has adjusted the expression pattern of various, mostly outer surface pr...

Carbonic anhydrase IX (CAIX) is a cancer-associated membrane protein frequently overexpressed in hypoxic solid tumours leading to enhanced tumour cell survival and invasion, and it has been proposed to be an attractive tumour-specific molecule for antibody-mediated targeting. This study aimed to generate a virus-like particle (VLP)-based CAIX vacci...

A model predictive control (MPC) method was investigated as a route to optimize and control the growth of E. coli BL21 (DE3) and biosynthesis of two different recombinant proteins (nerve growth factor NGF and coat protein of bacteriophage Qβ (Qβ-CP)). To determine the target trajectory for the E. coli cultivation process and estimate the model para...

Lyme disease affects several hundred thousand people worldwide annually, yet there is no registered vaccine for the disease available for human use. The disease is caused by Borrelia burgdorferi sensu lato complex bacteria, which harbor numerous outer surface proteins, and many of which have been targeted for vaccine development. However, to effect...

This scientific study explores the binding mechanisms of saccharine derivatives with human carbonic anhydrase IX (hCA IX), an antitumor drug target, with the aim of facilitating the design of potent and selective inhibitors. Through the use of crystallographic analysis, we investigate the structures of hCA IX - saccharine derivative complexes, unve...

Intraneuronal alpha-synuclein inclusions in the brain are hallmarks of so-called Lewy body diseases - Parkinson's disease and Dementia with Lewy bodies. Lewy bodies are cytoplasmic inclusions, containing mainly aggregated α-synuclein together with some other proteins including ubiquitin, neurofilament protein, and alpha B crystallin. In its monomer...

This scientific study explores the binding mechanisms of saccharine derivatives with human carbonic anhydrase IX (hCA IX), an antitumor drug target, with the aim of facilitating the design of potent and selective inhibitors. Through the use of crystallographic analysis, we investigate the structures of hCA IX ‐ saccharine derivative complexes, unve...

Bacterial microcompartments (BMCs) are prokaryotic organelles involved in several biochemical processes in bacterial cells. These cellular substructures consist of an icosahedral shell and an encapsulated enzymatic core. The outer shells of BMCs have been proposed as an attractive platform for the creation of novel nanomaterials, nanocages, and nan...

Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). Nuclear magnetic resonance studies show a Pro–VPg complex interaction and VPg tertiary structure; however, informat...

Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). NMR studies show a Pro-VPg complex interaction and VPg tertiary structure; however, information regarding structura...

The N-terminal domain (NT) of spider silk proteins (spidroins) is crucial for their storage at high concentrations, and also regulates silk assembly. NTs from the major ampullate (MaSp) and the minor ampullate (MiSp) spidroins are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH they dimerize to interconnect spidroins...

Endolysins are bacteriophage-encoded peptidoglycan-degrading enzymes with potential applications for treatment of multidrug-resistant bacterial infections. Hafnia phage Enc34 encodes an unusual endolysin with an N-terminal enzymatically active domain and a C-terminal transmembrane domain. The catalytic domain of the endolysin belongs to the conserv...

Among the twelve catalytically active carbonic anhydrase isozymes present in the human body, the CAIX is highly overexpressed in various solid tumors. The enzyme acidifies the tumor microenvironment enabling invasion and metastatic processes. Therefore, many attempts have been made to design chemical compounds that would exhibit high affinity and s...

MERS-CoV continues to cause human outbreaks, so far in 27 countries worldwide following the first registered epidemic in Saudi Arabia in 2012. In this study, we produced a nanovaccine based on virus-like particles (VLPs). VLPs are safe vaccine platforms as they lack any replication-competent genetic material, and are used since many years against h...

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses mRNA capping to evade the human immune system. The cap formation is performed by the SARS-CoV-2 mRNA cap methyltransferases (MTases) nsp14 and nsp16, which are emerging targets for the development of broad-spectrum antiviral agents. Here, we report results from high-throughput vi...

Site‐specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non‐enzymatic, post‐translational modification of N‐terminal HisTags. We report high‐yield, site‐selective in vitro α‐aminoacylation of peptides, glycoproteins, antibodies, and Virus‐like particles (VLPs) with azidogluconolactone at pH 7.5 in 1...

COVID-19 is a novel disease caused by SARS-CoV-2 which has conquered the world rapidly resulting in a pandemic that massively impacts our health, social activities, and economy. It is likely that vaccination is the only way to form “herd immunity” and restore the world to normal. Here we developed a vaccine candidate for COVID-19 based on the virus...

Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymatic processes, such as carbon fixation, choline, ethanolamine and propanediol degradation, and others. Formed of a semi‐permeable protein shell and an enzymatic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to...

Structure determination of adjuvant‐coupled antigens is essential for rational vaccine development but has so far been hampered by the relatively low antigen content in vaccine formulations and by their heterogeneous composition. Here we show that magic‐angle spinning (MAS) solid‐state NMR can be used to assess the structure of the influenza virus...

Structure determination of adjuvant‐coupled antigens is essential for rational vaccine development but has so far been hampered by the relatively low antigen content in vaccine formulations and by their heterogeneous composition. Here we show that magic‐angle spinning (MAS) solid‐state NMR can be used to assess the structure of the influenza virus...

BBA03 is a Borrelia burgdorferi outer surface lipoprotein encoded on one of the most conserved plasmids in Borrelia genome, linear plasmid 54 (lp54). Although many of its genes have been identified as contributing or essential for spirochete fitness in vivo, the majority of the proteins encoded on this plasmid have no known function and lack homolo...

Lyme disease is the most widespread vector-transmitted disease in North America and Europe, caused by infection with Borrelia burgdorferi sensu lato complex spirochetes. We report the solution NMR structure of the B. burgdorferi outer surface lipoprotein BBP28, a member of the multicopy lipoprotein (mlp) family. The structure comprises a tether pep...

In the design of high-affinity and enzyme isoform-selective inhibitors, we applied an approach of augmenting the substituents attached to the benzenesulfonamide scaffold in three ways, namely, substitutions at the 3,5- or 2,4,6-positions or expansion of the condensed ring system. The increased size of the substituents determined the spatial limitat...

The single-stranded RNA (ssRNA) bacteriophages are among the simplest known viruses with small genomes and exceptionally high mutation rates. The number of ssRNA phage isolates has remained very low, but recent metagenomic studies have uncovered an immense variety of distinct uncultured ssRNA phages. The coat proteins (CPs) in these genomes are par...

Virus-like particles (VLPs) can be used as efficient carriers of various antigens and therefore serve as attractive tools in vaccine development. Although VLPs of different viruses can be used, VLPs of ssRNA phages have convincing advantages due to their unique properties, including efficient protein production in bacterial and yeast expression sys...

ssRNA phages belonging to the family Leviviridae are among the tiniest viruses, infecting various Gram-negative bacteria by adsorption to their pilus structures. Due to their simplicity, they have been intensively studied as models for understanding various problems in molecular biology and virology. Several of the studied ssRNA characteristics, su...

This is the first book to systemize all levels of communicative behavior of phages. Phages represent the most diverse inhabitants on this planet. Until today they are completely underestimated in their number, skills and competences and still remain the dark matter of biology. Phages have serious effects on global energy and nutrient cycles. Phages...

Influenza, an acute, highly contagious respiratory disease, remains a significant threat to public health. More effective vaccination strategies aimed at inducing broad cross-protection not only against seasonal influenza variants, but also zoonotic and emerging pandemic influenza strains are urgently needed. A number of conserved protein targets t...

The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect LD...

A series of new 3-phenyl-5-aryl-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide derivatives was designed here, synthesized, and studied for carbonic anhydrase (CAs, EC 4.2.1.1) inhibitory activity against the human (h) isozymes I, II, and VII (cytosolic, off-target isoforms), and IX and XII (anticancer drug targets). Generally, CA I was...

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi sensu lato complex spirochetes. Through a complex enzootic cycle, the bacteria transfer between two different hosts: Ixodes ticks and mammalian organisms. At the start of the tick blood meal, the spirochetes located in the tick gut upregulate the expression of several genes, main...

Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC sh...

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi sensu lato complex spirochetes. The spirochete is located in the gut of the tick; as the infected tick starts the blood meal, the spirochete must travel through the hemolymph to the salivary glands, where it can spread to and infect the new host organism. In this study, we determ...

By applying an approach of a “ring with two tails”, a series of novel inhibitors possessing high-affinity and significant selectivity towards selected carbonic anhydrase (CA) isoforms has been designed. The “ring” consists of 2-chloro/bromo-benzenesulfonamide, where the sulfonamide group is as an anchor coordinating the Zn(II) in the active site of...

The periplasmic lipoprotein BB0365 of the Lyme disease agent Borrelia burgdorferi is expressed throughout mammalian infection and is essential for all phases of Lyme disease infection; its function, however, remains unknown. In the current study, our structural analysis of BB0365 revealed the same structural fold as that found in the NqrC and RnfG...

Lyme disease is an infection caused by the spirochete Borrelia burgdorferi after it is transmitted to a mammalian organism during a tick blood meal. B. burgdorferi encodes at least 140 lipoproteins located on the outer or inner membrane, thus facing the surroundings or the periplasmic space, respectively. However, most of the predicted lipoproteins...

Many viruses use polyprotein processing strategy to generate a large number of functional protein domains. gRNA of sobemoviruses code two polyproteins where a serine protease domain (Pro) is located at N-terminus of both precursors. Pro in sobemoviruses is active both in cis and in trans, but its activity in trans is mediated by naturally unfolded...

The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme borreliosis - the most common tick-borne disease in Europe and the United States. Spirochetes are transmitted from infected Ixodes ticks to the mammalian host when the ticks feed. In general, the transfer process of the borreliae is quite complicated, as the environments...

Background Protein shells assembled from viral coat proteins are an attractive platform for development of new vaccines and other tools such as targeted bioimaging and drug delivery agents. Virus-like particles (VLPs) derived from the single-stranded RNA (ssRNA) bacteriophage coat proteins (CPs) have been important and successful contenders in the...

Carbonic anhydrase IX (CA IX) is an isoform of CAs that is highly overexpressed in numerous human cancers and found essentially only in the stomach in healthy humans. Therefore, it is considered to be a target both for cancer therapy and cancer diagnostics. The CA IX is primarily found on the surface of cancer cells, it acidifies the environment an...

Long alpha helix (LAH) from influenza virus hemagglutinin (HA) stem or stalk domain is one of the most conserved influenza virus antigens. Expression of N-terminally extended LAH in E. coli leads to assembly of α-h elical homotrimer which is structurally nearly identical to the corresponding region of post-fusion form of native HA. This novel tri-s...

Purification of LAH-PP7 fusion protein. (A) Coomassie-stained PAAG illustrating individual purification steps. Lane 1, summary expression; lane 2, supernatant; lane 3, urea extract loaded onto Superdex column; lane 4, peak protein fraction from Superdex column. (B) Size-exclusion chromatography profile and (C) Electron microscopy indicating aggrega...

The ARRIVE guidelines checklist. (DOCX)

Full wwPDB X-ray structure validation report. (PDF)

The cross-reactivity of mouse sera. (A) Reactivity with group 1 HA proteins (H1, H2, H5, H9, H11 and H16). (B) Reactivity with group 2 HA proteins (H3, H4, H7, H10, H14 and H15). Mice were immunized with LAH-PP7 fusion protein or adjuvant alone (mock). The panels show the mean and standard deviation of the optical density at 405 nm of the sera from...

Comparison of HA tri-stalk ELISA results between SW and non-SW. HA tri-stalk specific seroreactivities were evaluated in pre-pandemic sera from 211 SW and 71 non-SW. (TIFF)

Comparison of Ab responses after vaccination of LAH-HBc VLPs [18] and tri-stalk protein. (TIFF)

Twenty-four novel sulfonamide derivatives incorporating dipeptide tails were synthesized by facile acylation reactions of homosulfanilamide through benzotriazole or dicyclohexyl carbodiimide (DCC) mediated coupling reactions. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against four human (h) isoform...

Borrelia (B.) bavariensis, B. burgdorferi, B. afzelii, B. garinii, B. spielmanii, and B. mayonii are the causative agents in Lyme disease. Lyme disease spirochetes reside in infected Ixodes ticks and are transferred to mammalian hosts during tick feeding. Once transmitted, spirochetes must overcome the first line of defense of the innate immune sys...

Bacteriophages of the Leviviridae family are small viruses with short single-stranded RNA (ssRNA) genomes. Protein-RNA interactions play a key role throughout the phage life cycle, and all of the conserved phage proteins - the maturation protein, the coat protein and the replicase - are able to recognize specific structures in the RNA genome. The p...

Human carbonic anhydrase (CA) IX has emerged as a promising anticancer target and a diagnostic biomarker for solid hypoxic tumors. Novel fluorinated CA IX inhibitors exhibited up to 50 pM affinity towards the recombinant human CA IX, selectivity over other CAs, and direct binding to Zn(II) in the active site of CA IX inducing novel conformational c...

2-Aminoquinazolin-4(3H)-ones were previously discovered as perspective leads for antimalarial drug development targeting the plasmepsins. Here we report the lead optimization studies with the aim to reduce inhibitor lipophilicity and increase selectivity versus the human aspartic protease Cathepsin D. Exploiting the solvent exposed area of the enzy...

Dynamic nuclear polarization (DNP) represents a powerful way to overcome the sensitivity limitation of magic‐angle spinning (MAS) NMR experiments. However, the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures, and in the presence of...

Dynamic nuclear polarization (DNP) represents a powerful way to overcome the sensitivity limitation of magic‐angle spinning (MAS) NMR experiments. However, the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures, and in the presence of...

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the most common tick-borne disease in the US and Europe. No potent human vaccine is currently available. The innate immune complement system is vital to host defense against pathogens, as complement activation on the surface of spirochetes results in bacterial killing. Comp...

Modern DNA sequencing capabilities have led to the discovery of a large number of new bacteriophage genomes, which are a rich source of novel proteins with an unidentified biological role. The genome of Enterobacter cancerogenus bacteriophage Enc34 contains several proteins of unknown function that are nevertheless conserved among distantly related...

Background The lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutin...

The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment. This article is pro...

H-detection in solid-state NMR of proteins has been traditionally combined with deuteration for both resolution and sensitivity reasons, with the optimal level of proton dilution being dependent on MAS rate. Here we present ¹H-detected ¹⁵N and ¹³C CP-HSQC spectra on two microcrystalline samples acquired at 60 and 111 kHz MAS and at ultra-high field...

A series of N-acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids (EMAC8000a-m) was designed and synthesised with the aim to target tumour associated carbonic anhydrase (CA) isoforms IX and XII. Most of the compounds were selective inhibitors of the tumour as-sociated CA XII. Moreover, resolution of EMAC8000d racemic mixture led to the isolatio...

A new chemotype with carbonic anhydrase (CA, EC 4.2.1.1) inhibitory action has been discovered, the homo-sulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) which have been designed considering the (sulfo)coumarins as lead molecules. An original synthetic strategy of a panel of such derivatives led to compounds with a unique inhibitory profile and...

A series of N-substituted saccharins incorporating aryl, alkyl and alkynyl moieties, as well as some ring opened derivatives were prepared and investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The widespread cytosolic isoforms CA I and II were not inhibited by these sulfonamides whereas transmembrane, tumor-associ...

Virions of the single-stranded RNA bacteriophages contain a single copy of the maturation protein which is bound to the phage genome and is required for infectivity of the particles. The maturation protein mediates the adsorption of the virion to bacterial pili and subsequent release and penetration of the genome into the host cell. Here, we report...

N(ε)-Trimethyllysine hydroxylase (TMLH) catalyses the first step in mammalian biosynthesis of carnitine, which plays a crucial role in fatty acid metabolism. The stereochemistry of the 3-hydroxy-N(ε)-trimethyllysine product of TMLH has not been defined. We report enzymatic and asymmetric synthetic studies, which define the product of TMLH catalysis...

Protein Structures G. Pintacuda et al. show in their Communication on page 15504 ff. that with 111 kHz magic-angle spinning probes, high-resolution ¹H-detected solid-state NMR spectra of proteins can be acquired without deuteration.

Festkörper-NMR-Spektroskopie Neue MAS-Sonden ermöglichen die hochauflösende NMR-Spektroskopie von unlöslichen, kristallinen oder selbstorganisierten Proteinaggregaten ohne Deuterierung. G. Pintacuda et al. beschreiben die Methode in der Zuschrift auf S. 15730 ff.

RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage...

Wir beschreiben die empfindliche Messung von α-Protonen in vollständig protonierten Proteinen durch Festkörper-NMR-Spektroskopie mit Rotation um den magischen Winkel (MAS) von 100–111 kHz. Die exzellente Auflösung in der Cα-Hα-Ebene wird für fünf Proteine gezeigt, darunter Mikrokristalle, ein sedimentierter Komplex, ein Kapsid und Amyloidfibrillen....

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100–111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα–Hα de...

A series of 5-substituted-benzylsulfanyl-thiophene-2-sulfonamides was prepared by reacting 5-bromo-thiophene-2-sulfonamide with 5-substituted-benzyl mercaptans. The new compounds were investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The cytosolic human (h) isoforms hCA I was poorly inhibited by the new sulfonamides (KIs in the range...

AP205 is a single-stranded RNA bacteriophage that has a coat protein sequence not similar to any other known ssRNA phage. Here, we report an atomic-resolution model of the AP205 virus-like particle based on a crystal structure of an unassembled coat protein dimer and a cryo-EM reconstruction of the assembled particle, together with secondary struct...

In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. Thi...

MS2 RNA view. (to Figure 2A) Proposed AP binding site RNA densities of nucleotides 3510-3527 (magenta) and 388-414 (blue) with fit in density (red) the secondary structure of the SL.

MS2 outside capsid view. (to Figure 1 A,B) The asymmetric reconstruction of bacteriophage MS2 showing the capsid (green-blue radially coloured) and the AP (yellow).

MS2 inside RNA view (to Figure 1 C, E) Inside bacteriophage MS2 the AP (yellow) is connected to a structured genome (grey) that is composed of stem loop structures, in which the double stranded helices can be observed.

View of RNA-interacting capsid dimers. (to Figure 2B) Asymmetric distribution of 23 capsid dimers (red) to which the density of the RNA SLs (grey) fit the translational operator.

Local resolution measurement. (to Supplementary figure 2) Resolution-coded colouring of the surface rendered RNA density.

Supplementary Figures 1-4

Plasmepsin IV from Plasmodium falciparum (PM IV) is a promising target for the development of novel antimalarial drugs. Here, the crystal structure of the truncated zymogen of PM IV (pPM IV), consisting of the mature enzyme plus a prosegment of 47 residues, has been determined at 1.5 Å resolution. pPM IV presents the fold previously described for s...

Significance Protein structure determination is key to the detailed description of many biological processes. The critical factor that would allow general application of magic-angle spinning (MAS) solid-state NMR to this end is improvement in sensitivity and resolution for as many nuclear spins as possible. This is achieved here with detection of r...

Aims The accumulation of long-chain fatty acids (FAs) and their CoA and carnitine esters is observed in the ischemic myocardium after acute ischemia-reperfusion. The aim of this study was to identify harmful FA intermediates and their detrimental mechanisms of action in mitochondria and the ischemic myocardium. Methods and Results In this study, we...

2-Aminoquinazolin-4(3H)-ones were identified as a novel class of malaria digestive vacuole plasmepsin inhibitors by using NMR-based fragment screening against Plm II. Initial fragment hit optimization led to a submicromolar inhibitor, which was co-crystallized with Plm II to produce an X-ray structure of the complex. The structure showed that 2-ami...

Plasmepsin II (PMII) is one of the ten plasmepsins (PMs) identified in the genome of Plasmodium falciparum , the causative agent of the most severe and deadliest form of malaria. Owing to the emergence of P. falciparum strains that are resistant to current antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is a constant pr...

Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered as a marker for cellular hypoxia, while it is not produced in most of the normal tis-sue. CA IX contributes to acidification of extracellular matrix, which in turn favors growth and metastasis of tumor. Therefore, CA IX is considered a promising anti-...