Towards a High-Affinity Peptidomimetic Targeting Proliferating Cell Nuclear Antigen from Aspergillus fumigatus

Abstract

Invasive fungal infections (IFIs) are prevalent in immunocompromised patients. Due to alarming levels of increasing resistance in clinical settings, new drugs targeting the major fungal pathogen Aspergillus fumigatus are required. Attractive drug targets are those involved in essential processes like DNA replication, such as proliferating cell nuclear antigens (PCNAs). PCNA has been previously studied in cancer research and presents a viable target for antifungals. Human PCNA interacts with the p21 protein, outcompeting binding proteins to halt DNA replication. The affinity of p21 for hPCNA has been shown to outcompete other associating proteins, presenting an attractive scaffold for peptidomimetic design. p21 has no A. fumigatus homolog to our knowledge, yet our group has previously demonstrated that human p21 can interact with A. fumigatus PCNA (afumPCNA). This suggests that a p21-based inhibitor could be designed to outcompete the native binding partners of afumPCNA to inhibit fungal growth. Here, we present an investigation of extensive structure-activity relationships between designed p21-based peptides and afumPCNA and the first crystal structure of a p21 peptide bound to afumPCNA, demonstrating that the A. fumigatus replication model uses a PIP-box sequence as the method for binding to afumPCNA. These results inform the new optimized secondary structure design of a potential peptidomimetic inhibitor of afumPCNA.

Keywords: Aspergillus fumigatus; DNA replication proteins; PCNA; X-ray crystallography; non-tag purification; peptide characterization.

Figure 1

PCNA homotrimer structure (PDB:7KQ0) [18]. (a) Trimer: PCNA surface shown in orange, IDCL shown in blue, and positively charged alpha helices shown in green. Central cavity is 35.4 Å wide. (b) Monomer: beta sheets shown in magenta and pink, alpha helices shown in green, and IDCL shown in blue. Made using Pymol Version 1.2 [19].

Figure 2

Sequence alignment of afumPCNA (top) (Uniprot: A0A0J5SJF1) vs. Human PCNA (bottom) (Uniprot: P38936). Red residues indicate hydrophobic residues, blue indicates negatively charged residues, pink indicates positively charged residues, and green indicates polar residues. Stars indicate conserved residues. Full stop “.” Indicates residues of shred characteristics. Indicates Semi colon “:” indicates residues of shared characteristics and structure.

Figure 3

afumPCNA bound with p21µ structure (PDB: 8GJF). (a) Trimer: afumPCNA surface shown in purple and p21µ shown in olive green. (b) Monomer: afumPCNA shown in purple and p21µ shown in olive green. (c) PIP-box binding site: 3₁₀ helical structure outlined in yellow and p21µ peptide shown in olive green. Made using Pymol [19]. Crystallographic information can be found in Table S2 and Figures S2 and S4.

Figure 4

Comparison of the p21µ peptide bound to hPCNA (PDB: 7KQ1) and p21µ peptide bound to afumPCNA (PDB: 8GJF). (a) hPCNA shown in grey, p21µ (PDB: 7KQ1) shown in salmon, and p21µ (PDB: 8GJF) shown in yellow. (b) Arg₁₄₃ and Glu₁₄₉ salt bridge interaction in p21µ (PDB: 8GJF) structure. (c) Conserved 3₁₀ helical and PIP-box region of p21µ peptides, residues 144–152: p21µ (PDB: 7KQ1) shown in salmon and p21µ (PDB: 8GJF) shown in yellow (RMSD: 0.939 Å).

Figure 5

afumPCNA bound with p21µ-afumRFC structure (PDB:8GJ5). (a) Trimer: afumPCNA surface shown in purple and p21µ-afumRFC shown in green. (b) Monomer: afumPCNA shown in purple and p21µ-afumRFC shown in green. (c) PIP-box binding site: 3₁₀ helical structure outlined in yellow and cyclical-like secondary structure outlined in orange. p21µ-afumRFC peptide shown in green. Made using PyMOL [19]. Crystallographic information can be found in Table S2 and Figures S3 and S5.

Figure 6

Previous p21-constrained peptides in comparison to the new p21μafumRFC peptide crystal structure. (a) p21μafumRFC peptide (green) crystal structure bound to the afumPCNA monomer (grey). p21μafumRFC peptide (green) produced an affinity of 295 ± 6.9 nM to hPCNA. (b) The Bimane peptide (purple) docked to the hPCNA monomer (grey), as shown in the computational model, produced an affinity of 570 ± 30 nM to hPCNA [38]. (c) p21μafumRFC peptide (green) superimposed over the Bimane peptide (purple) and hPCNA (grey) computational model. (d) p21μafumRFC peptide (green) superimposed over the Bimane peptide (purple) computational model.