Crystal structures of Kif2A complexed with WDR5 reveal the structural plasticity of WIN-S7 sites

Abstract

Chromosome congression and spindle assembly are essential for genomic stability and proper cell division, with deficiencies in these processes linked to tumorigenesis. WD repeat-containing protein 5 (WDR5), a core component of the mixed lineage leukemia (MLL) methyltransferase complex, directly binds to kinesin family member 2A (Kif2A) to regulate these mitotic events. Despite the importance of this interaction, its structural basis for Kif2A recognition by WDR5 remains unclear. Here, we determine the crystal structure of WDR5 in complex with a Kif2A-derived peptide (residues 114–122) at a resolution of 1.85 Å. Structural analysis reveals that Kif2A engages both the WIN and S7 sites of WDR5 via Arg117 and Ser121, with Ser121 forming hydrogen bonds with WDR5 Tyr191 and Lys259, driving Tyr191 rotation and opening the S7 pocket. Additional structures of WDR5 complexed with truncated or mutated Kif2A peptides and a WDR5 Y191F variant highlight the dynamic nature of Tyr191. Notably, anti-WDR5 compounds exhibit a similar binding mode at the WDR5 WIN-S7 site. The results of mutagenesis combined with isothermal titration calorimetry (ITC) assays underscore the critical roles of Arg117 and Ser121 in mediating the binding of Kif2A to WDR5. In summary, our findings provide atomic-level insights into the molecular mechanisms underlying the non-canonical mitotic function of the MLL/WDR5 complex and highlight WIN-S7 sites as promising therapeutic targets for diseases associated with chromosomal instability, such as cancers.

1. Introduction

Accurate chromosome segregation during mitosis is essential for maintaining genomic stability and preventing aneuploidy [1], a hallmark of many cancers [2,3]. Central to this process is the coordination of chromosome congression and spindle assembly, which are regulated by a network of protein interactions and posttranslational modifications [1,4]. Key players in this regulatory network include MLL complexes and kinesin motor proteins [5–7], both of which are essential for mitotic fidelity.

WDR5 is a highly conserved core component of MLL/SET1 methyltransferase complexes and is known for mediating histone H3 lysine 4 (H3K4) methylation, an epigenetic marker associated with active transcription [8–10]. This modification is critical for regulating gene expression during development and in response to cellular signals [11]. In addition to transcriptional regulation, WDR5 also functions as a scaffold protein, facilitating the assembly of diverse protein complexes through its WD40 repeat domain, which forms a β-propeller structure [6]. WDR5 has two distinct binding pockets: the WDR5-interacting (WIN) site and the WDR5-binding motif (WBM) site [6]. The WIN site accommodates various binding partners, including H3 [12–15], MLL proteins [16,17], MBD3C [18], KANSL1 [19], PDPK1 [20], LANA [21] and PTEN [22], whereas the WBM site interacts with proteins such as MYC [23], RBBP5 [24], and KANSL2 [19]. WDR5 is involved in diverse cellular processes, such as promoting cellular differentiation and bone formation [25] and maintaining stem cell pluripotency and self-renewal [26,27]. Given its multifaceted roles, particularly in cancer biology, where it is frequently overexpressed and linked to oncogenesis [28–30], WDR5 has emerged as an attractive drug target. Although the WIN site has been the primary focus of small-molecule inhibitor development [31,32], recent studies have identified the S7 pocket as an additional binding site that enhances inhibitor binding affinity [20,33–37]. This structural pocket was initially identified through studies with OICR-9429 [31,32], a small-molecule inhibitor that occupies a binding site formed by Phe133, Phe149, Pro173 and Tyr191, which is spatially distinct from the WIN site. Subsequent structural characterization by the Fesik group [34] led to the formal designation of this region as the S7 pocket, emphasizing its importance in structure-based inhibitor optimization. For example, dual-site inhibitors, such as compound 10, which targets both the WIN and S7 sites, have demonstrated potent on-target effects and preclinical efficacy in cancer models.

Emerging evidence suggests that the non-canonical role of WDR5 in mitosis is mediated by its interaction with Kif2A [7], a kinesin-13 family member that specializes in microtubule depolymerization. Kif2A plays a pivotal role in mitotic spindle assembly and chromosome segregation by regulating microtubule dynamics at plus ends, distinguishing it from cargo-transporting kinesins [38–40]. Structu...