Alexander Wlodawer’s research while affiliated with Frederick National Laboratory for Cancer Research, National Institutes of Health and other places

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Publications (504)


Advances in cryo-electron microscopy (cryoEM) for structure-based drug discovery
  • Literature Review

January 2025

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64 Reads

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Figure 1. Schematic representation of nucleoside metabolism in T. kodakarensis. Symbols: dR1P, deoxyribose 1-phosphate; dR5P, deoxyribose 5-phosphate; GA3P, glyceraldehyde 3-phosphate; AA, acetaldehyde; PNP, purine nucleoside phosphorylase; PPM Tk , phosphopentomutase from T. kodakarensis; DERA, deoxyribose-phosphate aldolase.
Figure 4. LC-MS analysis of purified PPM Tk . Mass spectrometry analysis of PPM Tk was performed after removal of His 6 -tag.
Figure 5. Cont.
Figure 7. Secondary structural analysis of PPM Tk by CD spectrometry. CD spectra were recorded from 260 to 200 nm in wavelength, at a temperature range of 30-90 • C. The image was created using GraphPad Prism ver. 7.04.
Figure 12. Superimposition of the protomers of experimentally determined PPM Tk (green), and AlphaFold2 predicted structure (tint). The alignment resulted in an RMSD of 0.466 Å.
Biophysical Characterization of a Novel Phosphopentomutase from the Hyperthermophilic Archaeon Thermococcus kodakarensis
  • Article
  • Full-text available

November 2024

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49 Reads

International Journal of Molecular Sciences

Phosphopentomutases catalyze the isomerization of ribose 1-phosphate and ribose 5-phosphate. Thermococcus kodakarensis, a hyperthermophilic archaeon, harbors a novel enzyme (PPMTk) that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity. Instead, PPMTk catalyzes the interconversion of ribose 1-phosphate and ribose 5-phosphate. Here, we report biophysical analysis, crystallization, and three-dimensional structure determination of PPMTk by X-ray diffraction at 2.39 Å resolution. The solved structure revealed a novel catalytic motif, unique to PPMTk, which makes this enzyme distinct from the homologous counterparts. We postulate that this novel catalytic motif may enable PPMTk to isomerize phosphopentose instead of phosphohexose. To the best of our knowledge, this is the first biophysical and structural analysis of a phosphopentomutase from hyperthermophilic archaea.

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Waterless structures in the Protein Data Bank

October 2024

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120 Reads

The absence of solvent molecules in high-resolution protein crystal structure models deposited in the Protein Data Bank (PDB) contradicts the fact that, for proteins crystallized from aqueous media, water molecules are always expected to bind to the protein surface, as well as to some sites in the protein interior. An analysis of the contents of the PDB indicated that the expected ratio of the number of water molecules to the number of amino-acid residues exceeds 1.5 in atomic resolution structures, decreasing to 0.25 at around 2.5 Å resolution. Nevertheless, almost 800 protein crystal structures determined at a resolution of 2.5 Å or higher are found in the current release of the PDB without any water molecules, whereas some other depositions have unusually low or high occupancies of modeled solvent. Detailed analysis of these depositions revealed that the lack of solvent molecules might be an indication of problems with either the diffraction data, the refinement protocol, the deposition process or a combination of these factors. It is postulated that problems with solvent structure should be flagged by the PDB and addressed by the depositors.


Structural and virologic mechanism of the emergence of resistance to Mpro inhibitors in SARS-CoV-2

September 2024

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25 Reads

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2 Citations

Proceedings of the National Academy of Sciences

We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (M pro ) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2 WK521 WT in VeroE6 TMPRSS2 cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2 WK521 E166V ), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2 E166V was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of M pro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1’-Glu166 interactions. TKB245 stayed bound to M pro E166V , whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of M pro , and compromised the enzymatic activity; the Ki values of recombinant M pro E166V for nirmatrelvir and TKB245 were 117±3 and 17.1±1.9 µM, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to M pro E166V than nirmatrelvir. SARS-CoV-2 WK521 WT selected with 5h acquired A191T substitution in M pro (SARS-CoV-2 WK521 A191T ) and better replicated in the presence of 5h, than SARS-CoV-2 WK521 WT . However, no significant enzymatic or structural changes in M pro A191T were observed. The replicability of SARS-CoV-2 WK521 E166V proved to be compromised compared to SARS-CoV-2 WK521 WT but predominated over SARS-CoV-2 WK521 WT in the presence of nirmatrelvir. The replicability of SARS-CoV-2 WK521 A191T surpassed that of SARS-CoV-2 WK521 WT in the absence of 5h, confirming that A191T confers enhanced viral fitness. The present data should shed light on the understanding of the mechanism of SARS-CoV-2’s drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.



Selected statistics of the three models of Type III antifreeze protein isoform HPLC12.
In some cases more complicated approaches to refinement of macromolecular structures are not necessary

June 2024

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15 Reads

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1 Citation

The manuscript ‘Modeling a unit cell: crystallographic refinement procedure using the biomolecular MD simulation platform Amber’ presents a novel protein structure refinement method claimed to offer improvements over traditional techniques like Refmac5 and Phenix. Our re-evaluation suggests that while the new method provides improvements, traditional methods achieve comparable results with less computational effort.


Towards a dependable data set of structures for L -asparaginase research

June 2024

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40 Reads

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3 Citations

The Protein Data Bank (PDB) includes a carefully curated treasury of experimentally derived structural data on biological macromolecules and their various complexes. Such information is fundamental for a multitude of projects that involve large-scale data mining and/or detailed evaluation of individual structures of importance to chemistry, biology and, most of all, to medicine, where it provides the foundation for structure-based drug discovery. However, despite extensive validation mechanisms, it is almost inevitable that among the ∼215 000 entries there will occasionally be suboptimal or incorrect structure models. It is thus vital to apply careful verification procedures to those segments of the PDB that are of direct medicinal interest. Here, such an analysis was carried out for crystallographic models of L-asparaginases, enzymes that include approved drugs for the treatment of certain types of leukemia. The focus was on the adherence of the atomic coordinates to the rules of stereochemistry and their agreement with the experimental electron-density maps. Whereas the current clinical application of L-asparaginases is limited to two bacterial proteins and their chemical modifications, the field of investigations of such enzymes has expanded tremendously in recent years with the discovery of three entirely different structural classes and with numerous reports, not always quite reliable, of the anticancer properties of L-asparaginases of different origins.


P1' specificity of the S219V/R203G mutant tobacco etch virus protease

April 2024

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19 Reads

Proteins Structure Function and Bioinformatics

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1′ autoproteolytic cleavage‐resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self‐inactivation, but also exhibited greater stability and catalytic efficiency than the wild‐type enzyme. An R203G substitution has been reported to further relax the P1′ specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1′ specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1′ amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1′ variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme–substrate interactions were analyzed in silico. The results indicate highly similar P1′ preferences for both enzymes, many side‐chains can be accommodated by the S1′ binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.


RrA, an enzyme from Rhodospirillum rubrum, is a prototype of a new family of short‐chain L‐asparaginases

March 2024

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54 Reads

L‐Asparaginases (ASNases) catalyze the hydrolysis of L‐Asn to L‐Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300–400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum, reported here and referred to as RrA, consists of only 172 amino acid residues. RrA is homologous to the N‐terminal domain of typical bacterial class 1 ASNases and exhibits millimolar affinity for L‐Asn. In this study, we demonstrate that RrA belongs to a unique family of cytoplasmic, short‐chain ASNases (scASNases). These proteins occupy a distinct region in the sequence space, separate from the regions typically assigned to class 1 ASNases. The scASNases are present in approximately 7% of eubacterial species, spanning diverse bacterial lineages. They seem to be significantly enriched in species that encode for more than one class 1 ASNase. Here, we report biochemical, biophysical, and structural properties of RrA, a member of scASNases family. Crystal structures of the wild‐type RrA, both with and without bound L‐Asp, as well as structures of several RrA mutants, reveal topologically unique tetramers. Moreover, the active site of one protomer is complemented by two residues (Tyr21 and Asn26) from another protomer. Upon closer inspection, these findings clearly outline scASNases as a stand‐alone subfamily of ASNases that can catalyze the hydrolysis of L‐Asn to L‐Asp despite the lack of the C‐terminal domain that is present in all ASNases described structurally to date.


Citations (59)


... It may also explain the synergy observed between the L50F and the E166A/L167F mutations. Specifically, L50F improved the activity of the E166A/L167F mutant by 6-fold in the L50F/E166A/L167F triple mutant, in comparison to the 3-fold activity difference between the L50F single mutant and WT 28 . ...

Reference:

Distal protein-protein interactions contribute to nirmatrelvir resistance
Structural and virologic mechanism of the emergence of resistance to Mpro inhibitors in SARS-CoV-2
  • Citing Article
  • September 2024

Proceedings of the National Academy of Sciences

... PET has been extensively applied to production and manufacturing of beverage bottles. At present, the recovery rate of beverage bottles in developing countries is only 94 %, which is considerably lower than those in developed countries in Europe and America [1,2]. As shown in Fig. 1, common beverage bottles are composed of bottle body (PET material) and label paper (Biaxially oriented polypropylene film -BOPP material). ...

Development of Enzyme-Based Approaches for Recycling PET on an Industrial Scale
  • Citing Article
  • January 2024

Biochemistry

... The Alphavirus genus can be further divided into the New and Old World alphaviruses. Among these, the New World viruses include the Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis virus (WEEV), while the Old World virus group includes Sindbis virus (SINV), Semliki Forest virus (SFV), and the Chikungunya virus (CHIKV) [19,20]. The VEEV particles are 60-70 nm in diameter and the nucleocapsid is 30-35 nm in diameter, hexagonal in shape, when negatively stained [21] (Fig. 2A). ...

Self-inhibited State of Venezuelan Equine Encephalitis Virus (VEEV) nsP2 Cysteine Protease: A Crystallographic and Molecular Dynamics Analysis

Journal of Molecular Biology

... One common feature of the GP family of proteases is that their catalytic residue includes a glutamic acid and an additional variable amino acid [20]. GPs are involved in various biological functions, such as acting as plant and bacterial pathogens and playing essential roles in several diseases, including celiac disease and cancer. ...

Overview of the Properties of Glutamic Peptidases That Are Present in Plant and Bacterial Pathogens and Play a Role in Celiac Disease and Cancer
  • Citing Article
  • January 2023

Biochemistry

... These findings are also consistent with previously reported eQTL results for the haplotype block linked to rs4294451, where the T allele was associated with higher levels of DPYD expression in human liver specimens (Etheridge et al., 2020), and our observation that the T allele is associated with cellular resistance to 5-FU ( Figure 4E). The binding of CEBPB to specific motifs has recently been shown to be cell-type specific and to rely on non-consensus binding motifs with binding strengths that can be modulated by the sequence and structure of surrounding DNA regions (Cohen et al., 2018;Lountos et al., 2022), providing a possible explanation for the discrepancy between predicted and observed results. ...

Structural basis for cell type specific DNA binding of C/EBPβ: The case of cell cycle inhibitor p15INK4b promoter

Journal of Structural Biology

... It should be noted that psychrotolerant strains At. ferrooxidans PG05 and MC2.2 [68], presented up to 4 gene copies, one of which is a truncated form that lacks the ATPase and proteolytic domains. This truncated form of Lon protease has previously been shown to be present in other microorganisms and to be active in foldase activity [69]. On the other hand, no Lon protease gene was detected in representatives of the phylum Actinomycetota. ...

Unique Structural Fold of LonBA Protease from Bacillus subtilis, a Member of a Newly Identified Subfamily of Lon Proteases

International Journal of Molecular Sciences

... Relative activities are presented in Table 3. Similar to the situation found in typical class 1 ASNases, mutation of the catalytic Lys residue (K158M in RrA) completely inactivates the enzyme Strzelczyk et al., 2020;Strzelczyk et al., 2023) (Table 3). Another mutated residue was Tyr21. ...

The dimeric form of bacterial l‐asparaginase YpAI is fully active

... This lack of methods for enrichment of tyrosine glycosylation could be the main limiting factor for application of LC-MS/MS characterization. In fact, the development and application of antibodies for tyrosine O-GalNAcylation recently indicated that tyrosine glycosylation is widely expressed in most human tissues and is likely a ubiquitous and underappreciated PTM [40]. ...

Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O -GalNAcylation in Numerous Human Tissues and Cell Lines
  • Citing Article
  • September 2022

Journal of the American Chemical Society

... The first step of the viral infection is the union of the spike protein (S) of the virus with (subunit S1) the angiotensin converting enzyme 2 (ACE2) which contributes to a conformational change in the S2 subunit of the S protein resulting in the binding of the virus with the trans membrane protein serine protease 2 (TMPRSS2). Both the 2 TMPRSS2 and ACE2 are present in the lipid rafts that are microdomains in the cell membrane of the host [2]. SARS-CoV-2 may use two mechanisms for its internalization: 1) fusion and 2) endocytosis. ...

Structural investigations of proteins encoded by SARS‐CoV ‐2

... Derewenda (2023) recently wrote a comprehensive review on the role of weak hydrogen bonds in the structures of proteins and nucleic acids [7]. Independently, it was also reported that subtilisin-like superfamily proteins [8,9], which belong to clan SB serine peptidases [3], also have a similar 3D arrangement of four catalytic residues mentioned above [5,6], which we will refer to as the "catalytic tetrad" of serine proteases when specifically speaking about the three aforementioned superfamilies. As opposed to trypsin-like serine proteases, the alpha/beta-hydrolases have five key residues (the catalytic triad plus two) to carry out protein function, which we, therefore, call a "catalytic pentad" [10]. ...

Serine-Carboxyl Peptidases, Sedolisins: From Discovery to Evolution

Biochemistry