Structural mechanism of a drug-binding process involving a large conformational change of the protein target.

Proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of such events have been elusive. Here, we report unguided molecular dynamics simulations of Abl kinase binding to the cancer drug imatinib. In the simulations, imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein to reach a bound complex that closely resembles published crystal structures. Moreover, the simulations reveal a surprising local structural instability in the C-terminal lobe of Abl kinase during binding. The unstable region includes a number of residues that, when mutated, confer imatinib resistance by an unknown mechanism. Based on the simulations, NMR spectra, hydrogen-deuterium exchange measurements, and thermostability measurements and estimates, we suggest that these mutations confer imatinib resistance by exacerbating structural instability in the C-terminal lobe, rendering the imatinib-bound state energetically unfavorable.

Domain Architecture of the Nonreceptor Tyrosine Kinase Ack1.

The nonreceptor tyrosine kinase (NRTK) Ack1 comprises a distinct arrangement of non-catalytic modules. Its SH3 domain has a C-terminal to the kinase domain (SH1), in contrast to the typical SH3-SH2-SH1 layout in NRTKs. The Ack1 is the only protein that shares a region of high homology to the tumor suppressor protein Mig6, a modulator of EGFR. The vertebrate Acks make up the only tyrosine kinase (TK) family known to carry a UBA domain. The GTPase binding and SAM domains are also uncommon in the NRTKs. In addition to being a downstream effector of receptor tyrosine kinases (RTKs) and integrins, Ack1 can act as an epigenetic regulator, modulate the degradation of the epidermal growth factor receptor (EGFR), confer drug resistance, and mediate the progression of hormone-sensitive tumors. In this review, we discuss the domain architecture of Ack1 in relation to other protein kinases that possess such defined regulatory domains.

Biochemical Studies of Systemic Lupus Erythematosus-Associated Mutations in Nonreceptor Tyrosine Kinases Ack1 and Brk.

Tyrosine kinases (TKs) play essential roles in signaling processes that regulate cell survival, migration, and proliferation. Dysregulation of tyrosine kinases underlies many disorders, including cancer, cardiovascular and developmental diseases, as well as pathologies of the immune system. Ack1 and Brk are nonreceptor tyrosine kinases (NRTKs) best known for their roles in cancer. Here, we have biochemically characterized novel Ack1 and Brk mutations identified in patients with systemic lupus erythematosus (SLE). These mutations are the first SLE-linked polymorphisms found among NRTKs. We show that two of the mutants are catalytically inactive, while the other three have reduced activity. To understand the structural changes associated with the loss-of-function phenotype, we solved the crystal structure of one of the Ack1 kinase mutants, K161Q. Furthermore, two of the mutated residues (Ack1 A156 and K161) critical for catalytic activity are highly conserved among other TKs, and their substitution in other members of the kinase family could have implications in cancer. In contrast to canonical gain-of-function mutations in TKs observed in many cancers, we report loss-of-function mutations in Ack1 and Brk, highlighting the complexity of TK involvement in human diseases.

Allosteric regulation and inhibition of protein kinases.

The human genome encodes more than 500 different protein kinases: signaling enzymes with tightly regulated activity. Enzymatic activity within the conserved kinase domain is influenced by numerous regulatory inputs including the binding of regulatory domains, substrates, and the effect of post-translational modifications such as autophosphorylation. Integration of these diverse inputs occurs via allosteric sites that relate signals via networks of amino acid residues to the active site and ensures controlled phosphorylation of kinase substrates. Here, we review mechanisms of allosteric regulation of protein kinases and recent advances in the field.

IFITM proteins assist cellular uptake of diverse linked chemotypes.

The search for cell-permeable drugs has conventionally focused on low-molecular weight (MW), nonpolar, rigid chemical structures. However, emerging therapeutic strategies break traditional drug design rules by employing flexibly linked chemical entities composed of more than one ligand. Using complementary genome-scale chemical-genetic approaches we identified an endogenous chemical uptake pathway involving interferon-induced transmembrane proteins (IFITMs) that modulates the cell permeability of a prototypical biopic inhibitor of MTOR (RapaLink-1, MW: 1784 g/mol). We devised additional linked inhibitors targeting BCR-ABL1 (DasatiLink-1, MW: 1518 g/mol) and EIF4A1 (BisRoc-1, MW: 1466 g/mol), uptake of which was facilitated by IFITMs. We also found that IFITMs moderately assisted some proteolysis-targeting chimeras and examined the physicochemical requirements for involvement of this uptake pathway.

Extended DNA-binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks.

Elevated DNA replication stress causes instability of the DNA replication fork and increased DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator silencing-defective 2 (SDE2) is known to bind to TIMELESS (TIM), a protein of the fork protection complex, and enhances its stability, thereby supporting replisome activity at DNA replication forks. However, the DNA-binding activity of SDE2 is not well defined. Here, we structurally and functionally characterize a new conserved DNA-binding motif related to the SAP (SAF-A/B, Acinus, PIAS) domain in human SDE2 and establish its preference for ssDNA. Our NMR solution structure of the SDE2SAP domain reveals a helix-extended loop-helix core with the helices aligned parallel to each other, consistent with known canonical SAP folds. Notably, we have shown that the DNA interaction of this SAP domain extends beyond the core SAP domain and is augmented by two lysine residues in the C-terminal tail, which is uniquely positioned adjacent to the SAP motif and conserved in the pre-mRNA splicing factor SF3A3. Furthermore, we found that mutation in the SAP domain and extended C terminus not only disrupts ssDNA binding but also impairs TIM localization at replication forks, thus inhibiting efficient fork progression. Taken together, our results establish SDE2SAP as an essential element for SDE2 to exert its role in preserving replication fork integrity via fork protection complex regulation and highlight the structural diversity of the DNA-protein interactions achieved by a specialized DNA-binding motif.

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations.

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often "cryptic" without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein-ligand systems.

Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance.

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type-like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway.

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity.

KA1 Domains: Unity in Mechanistic Diversity.

The enzymatic activity of protein kinases is often tightly controlled by regulatory domains with a conserved structural mechanism. In this issue of Structure, Emptage et al. (2018) report how the kinase associated-1 domain (KA1) autoinhibits kinase activity with a striking structural diversity.