Structural and biochemical studies of human proliferating cell nuclear antigen complexes provide a rationale for cyclin association and inhibitor design

Abstract

The interactions between the tumor suppressor protein p21 WAF1 and the cyclin-dependent kinase (CDK) complexes and with proliferating cell nuclear antigen (PCNA) regulate and coordinate the processes of cell-cycle progression and DNA replication. We present the x-ray crystal structure of PCNA complexed with a 16-mer peptide related to p21 that binds with a Kd of 100 nM. Two additional crystal structures of native PCNA provide previously undescribed structures of uncomplexed human PCNA and show that significant changes on ligand binding include rigidification of a number of flexible regions on the surface of PCNA. In the competitive binding experiments described here, we show that a 20-mer sequence from p21 can be associated simultaneously with PCNA and CDK͞cyclin complexes. A structural model for this quaternary complex is presented in which the C-terminal sequence of p21 acts like double-sided tape and docks to both the PCNA and cyclin molecules. The quaternary complex shows little direct interaction between PCNA and cyclin, giving p21 the role of an adaptor molecule. Taken together, the biochemical and structural results delineate a druggable inhibitor site on the surface of PCNA that may be exploited in the design of peptidomimetics, which will act independently of cyclin-groove inhibitors.p21 ͉ x-ray ͉ structure ͉ protein ͉ interaction P roliferating cell nuclear antigen (PCNA) is an essential auxiliary protein for the processes of both DNA replication and repair. It stimulates the activity of DNA polymerase ␦ and increases its processivity by acting as a clamp platform that slides along the DNA template (1-3). Apart from polymerase ␦, PCNA associates with a host of other proteins, either involved directly in DNA replication and repair or in the regulation of these processes (4). The presence of a common PCNA-binding motif in such proteins suggests that regulation may depend largely on PCNA partner proteins competing with one another for access to PCNA.Deregulation of PCNA expression is a hallmark of many proliferative diseases, and in the clinic PCNA serves as a general proliferative marker, especially in the prognosis of tumor development (5). In fact, PCNA expression levels are directly related to the malignancy of various tumors, and antisense oligonucleotidemediated suppression of PCNA expression was demonstrated to selectively inhibit gastric cancer cell proliferation in vitro and in vivo (6). Antisense strategies targeting PCNA mRNA also have shown promise in models of other proliferative diseases, including glomerular nephritis (7) and rheumatoid arthritis (8). The fact that PCNA is required absolutely for cell proliferation indicates that pharmacological modulation of PCNA function should not be able to be circumvented by compensatory pathways. Furthermore, the ablation of PCNA expression or function in cells under proliferative stimuli appears to constitute an apoptotic trigger (5), suggesting that effective elimination of hyperproliferative cells should be possible in a therapeutic setting.The tum...

Deviating the level of proliferating cell nuclear antigen inTrypanosoma bruceielicits distinct mechanisms for inhibiting proliferation and cell cycle progression

“…This has fundamentally restricted the types of studies for chemotherapeutic interventions of PCNA to human proliferative diseases such as cancer, arthritis and nephritis. 52,[56][57][58][59] The results of this study validate TbPCNA as a viable target for therapeutic intervention against African trypanosomiasis and broaden the categories of where PCNA therapeutics may be beneficial to infectious disease research. Future studies will elucidate mechanisms of TbPCNA regulation in T. brucei.…”

Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesis

“…To understand the molecular basis by which PCNA phosphorylation blocks its interactions with MutSα and MutSβ, virtual protein structure modeling of PCNA-Y211F, PCNA-Y211D, and PCNA-Y211E was performed, using the published crystal structure of PCNA as a starting point (35,36). Y211 is located in the A2 α-helix and oriented to the central loop (residues 41-45), which is underneath the hydrophobic pocket.…”

Increased islet neogenesis without increased islet mass precedes autoimmune attack in diabetes-prone rats

“…Cyclin/CDK and PCNA form a quaternary complex which regulates cell cycle progression. 59 CyclinD2, the dominant D-type cyclin in adult islets, 45 is an essential b-cell replication factor implicated in triggering b-cell entry into the cell cycle 60,61 and regulating b-cell mass. 57 Cdkn1a inhibits CDKs, promoting G1 cell cycle arrest 31 and blocking islet replication in response to growth factors.…”