Abstract
The presence of protein structures with atypical folds in the Protein Data Bank (PDB) is rare and may result from naturally occurring knots or crystallographic errors. Proper characterisation of such folds is imperative to understanding the basis of naturally existing knots and correcting crystallographic errors. If left uncorrected, such errors can frustrate downstream experiments that depend on the structures containing them. An atypical fold has been identified in P. falciparum dihydrofolate reductase (PfDHFR) between residues 20–51 (loop 1) and residues 191–205 (loop 2). This enzyme is key to drug discovery efforts in the parasite, necessitating a thorough characterisation of these folds. Using multiple sequence alignments (MSA), a unique insert was identified in loop 1 that exacerbates the appearance of the atypical fold-giving it a slipknot-like topology. However, PfDHFR has not been deposited in the knotted proteins database, and processing its structure failed to identify any knots within its folds. The application of protein homology modelling and molecular dynamics simulations on the DHFR domain of P. falciparum and those of two other organisms (E. coli and M. tuberculosis) that were used as molecular replacement templates in solving the PfDHFR structure revealed plausible unentangled or open conformations of these loops. These results will serve as guides for crystallographic experiments to provide further insights into the atypical folds identified.
Highlights
Information on the three-dimensional (3D) structure composition of biological macromolecules— proteins and nucleic acids—and their associated ligands and cofactors are archived and managed by the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) [1,2]
We identified a slipknot-like topology formed between the active site lid-loop and a second loop of the Plasmodium falciparum DHFR (Pf DHFR) enzyme (Figure 1)
An atypical fold was identified in the P. falciparum dihydrofolate reductase (Pf DHFR) structure, involving residues 20–51 and 191–205
Summary
Information on the three-dimensional (3D) structure composition of biological macromolecules— proteins and nucleic acids—and their associated ligands and cofactors are archived and managed by the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) [1,2]. Among the over 160 thousand protein structures currently deposited in the RCSB PDB is a subset of rare proteins with entangled or knotted topologies within their structural folds [3]. Different structural entanglements have been identified, and the protein structures concerned (referred to as knotted proteins) are deposited in the KnotProt database [4,5]. Some of the entangled protein topologies identified include Slipknots (Figure 1), which are knotted subchains that appear unentangled as a whole; probabilistic and deterministic knots including disulfide or ion interactions; cystine knots; and knotoides [5]. The KnotProt database performs regular self-updates, where new PDB entries are scanned for novel entangled topologies. The database hosts just over 2000 knotted protein entries, more than half of which are slipknots [5]. The exact role of protein structural entanglements is unknown, they have been suggested to play important roles in the active site shaping and improvement in overall thermal stability of the protein [3,6]
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