Structure of the sliding clamp from the fungal pathogen Aspergillus fumigatus (AfumPCNA) and interactions with Human p21

• Published in: The FEBS journal 2017-03, Vol.284 (6), p.985-1002
• Main Authors: Marshall, Andrew C., Kroker, Alice J., Murray, Lauren A.M., Gronthos, Kahlia, Rajapaksha, Harinda, Wegener, Kate L., Bruning, John B.
• Format: Article
• Language: English
• Subjects: Affinity; Amino Acid Motifs - genetics; Antigens; Aspergillosis - genetics; Aspergillosis - pathology; Aspergillus; Aspergillus fumigatus; Aspergillus fumigatus - chemistry; Atomic structure; Binding Sites; Clamps; Crystal structure; Crystallography, X-Ray; Cyclin-dependent kinase inhibitor p21; Cyclin-Dependent Kinase Inhibitor p21 - chemistry; Cyclin-Dependent Kinase Inhibitor p21 - genetics; Data banks; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA biosynthesis; DNA Replication - genetics; Fluorescence; Fluorescence polarization; Fungal infections; Fungi; GTP-binding protein; Host-Pathogen Interactions - genetics; Humans; Hydrophobicity; Microorganisms; Molecular biology; Molecular dynamics; p21; Pathogens; PCNA; PIP‐box; Proliferating cell nuclear antigen; Proliferating Cell Nuclear Antigen - chemistry; Proliferating Cell Nuclear Antigen - genetics; Protein Binding; Protein Interaction Domains and Motifs - genetics; Protein structure; Protein Structure, Secondary; Proteins; Replication; Secondary structure; Sliding; sliding clamp; Yard waste
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.14035

The fungal pathogen Aspergillus fumigatus has been implicated in a drastic increase in life‐threatening infections over the past decade. However, compared to other microbial pathogens, little is known about the essential molecular processes of this organism. One such fundamental process is DNA replication. The protein responsible for ensuring processive DNA replication is PCNA (proliferating cell nuclear antigen, also known as the sliding clamp), which clamps the replicative polymerase to DNA. Here we present the first crystal structure of a sliding clamp from a pathogenic fungus (A. fumigatus), at 2.6Å. Surprisingly, the structure bears more similarity to the human sliding clamp than other available fungal sliding clamps. Reflecting this, fluorescence polarization experiments demonstrated that AfumPCNA interacts with the PCNA‐interacting protein (PIP‐box) motif of human p21 with an affinity (Kd) of 3.1 μm. Molecular dynamics simulations were carried out to better understand how AfumPCNA interacts with human p21. These simulations revealed that the PIP‐box bound to AfuPCNA forms a secondary structure similar to that observed in the human complex, with a central 310 helix contacting the hydrophobic surface pocket of AfumPCNA as well as a β‐strand that forms an antiparallel sheet with the AfumPCNA surface. Differences in the 310 helix interaction with PCNA, attributed to residue Thr131 of AfumPCNA, and a less stable β‐strand formation, attributed to residues Gln123 and His125 of AfumPCNA, are likely causes of the over 10‐fold lower affinity of the p21 PIP‐box for AfumPCNA as compared to hPCNA. Database The atomic coordinates and structure factors for the Aspergillus fumigatus sliding clamp can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession code 5TUP. We present the 2.6 Å structure of proliferating cell nuclear antigen (PCNA) from the pathogenic fungus, Aspergillus fumigatus, and demonstrate its high‐affinity interaction with a small peptide derived from host protein p21. Comparison with the human PCNA structure, coupled with MD simulations, highlight features that may contribute to differences in binding affinity. This knowledge will be useful for future antifungal design.