Molecular basis for antibody recognition of multiple drug-peptide/MHC complexes.

The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.

The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability.

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of ß-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.

Exploring switch II pocket conformation of KRAS(G12D) with mutant-selective monobody inhibitors.

The G12D mutation is among the most common KRAS mutations associated with cancer, in particular, pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over the G12D mutation in HRAS and NRAS. Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and α3 helix, and captured this pocket in the most widely open form reported to date. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of KRAS Asp12, a feature that closely resembles that of a small-molecule inhibitor, MTRX1133. The monobody also directly interacted with H95, a residue not conserved in RAS isoforms. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a monobody generated hundreds of functional and nonfunctional single-point mutants, which identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results further illustrate the plasticity of the S-II pocket, which may be exploited for the design of next-generation KRAS(G12D)-selective inhibitors.

Monobody Inhibitor Selective to the Phosphatase Domain of SHP2 and its Use as a Probe for Quantifying SHP2 Allosteric Regulation.

SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the "PTP-dead" C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.

Creating MHC-Restricted Neoantigens with Covalent Inhibitors That Can Be Targeted by Immune Therapy.

Intracellular oncoproteins can be inhibited with targeted therapy, but responses are not durable. Immune therapies can be curative, but most oncogene-driven tumors are unresponsive to these agents. Fragments of intracellular oncoproteins can act as neoantigens presented by the major histocompatibility complex (MHC), but recognizing minimal differences between oncoproteins and their normal counterparts is challenging. We have established a platform technology that exploits hapten-peptide conjugates generated by covalent inhibitors to create distinct neoantigens that selectively mark cancer cells. Using the FDA-approved covalent inhibitors sotorasib and osimertinib, we developed "HapImmune" antibodies that bind to drug-peptide conjugate/MHC complexes but not to the free drugs. A HapImmune-based bispecific T-cell engager selectively and potently kills sotorasib-resistant lung cancer cells upon sotorasib treatment. Notably, it is effective against KRASG12C-mutant cells with different HLA supertypes, HLA-A*02 and A*03/11, suggesting loosening of MHC restriction. Our strategy creates targetable neoantigens by design, unifying targeted and immune therapies. SIGNIFICANCE: Targeted therapies against oncoproteins often have dramatic initial efficacy but lack durability. Immunotherapies can be curative, yet most tumors fail to respond. We developed a generalizable technology platform that exploits hapten-peptides generated by covalent inhibitors as neoantigens presented on MHC to enable engineered antibodies to selectively kill drug-resistant cancer cells. See related commentary by Cox et al., p. 19. This article is highlighted in the In This Issue feature, p. 1.

The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death.

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.

Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS monobody targeting the Switch I/II pocket.

RAS mutants are major therapeutic targets in oncology with few efficacious direct inhibitors available. The identification of a shallow pocket near the Switch II region on RAS has led to the development of small-molecule drugs that target this site and inhibit KRAS(G12C) and KRAS(G12D). To discover other regions on RAS that may be targeted for inhibition, we have employed small synthetic binding proteins termed monobodies that have a strong propensity to bind to functional sites on a target protein. Here, we report a pan-RAS monobody, termed JAM20, that bound to all RAS isoforms with nanomolar affinity and demonstrated limited nucleotide-state specificity. Upon intracellular expression, JAM20 potently inhibited signaling mediated by all RAS isoforms and reduced oncogenic RAS-mediated tumorigenesis in vivo. NMR and mutation analysis determined that JAM20 bound to a pocket between Switch I and II, which is similarly targeted by low-affinity, small-molecule inhibitors, such as BI-2852, whose in vivo efficacy has not been demonstrated. Furthermore, JAM20 directly competed with both the RAF(RBD) and BI-2852. These results provide direct validation of targeting the Switch I/II pocket for inhibiting RAS-driven tumorigenesis. More generally, these results demonstrate the utility of tool biologics as probes for discovering and validating druggable sites on challenging targets.

Identification of the nucleotide-free state as a therapeutic vulnerability for inhibition of selected oncogenic RAS mutants.

RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

Targeting the KRAS α4-α5 allosteric interface inhibits pancreatic cancer tumorigenesis.

RAS is the most frequently mutated oncogene in human cancer with nearly ~20% of cancer patients possessing mutations in one of three RAS genes (K, N or HRAS). However, KRAS is mutated in nearly 90% of pancreatic ductal carcinomas (PDAC). Although pharmacological inhibition of RAS has been challenging, KRAS(G12C)-specific inhibitors have recently entered the clinic. While KRAS(G12C) is frequently expressed in lung cancers, it is rare in PDAC. Thus, more broadly efficacious RAS inhibitors are needed for treating KRAS mutant-driven cancers such as PDAC. A RAS-specific tool biologic, NS1 Monobody, inhibits HRAS- and KRAS-mediated signalling and oncogenic transformation both in vitro and in vivo by targeting the α4-α5 allosteric site of RAS and blocking RAS self-association. Here, we evaluated the efficacy of targeting the α4-α5 interface of KRAS as an approach to inhibit PDAC development using an immunocompetent orthotopic mouse model. Chemically regulated NS1 expression inhibited ERK and AKT activation in KRAS(G12D) mutant KPC PDAC cells and reduced the formation and progression of pancreatic tumours. NS1-expressing tumours were characterized by increased infiltration of CD4 + T helper cells. These results suggest that targeting the #x3B1;4-#x3B1;5 allosteric site of KRAS may represent a viable therapeutic approach for inhibiting KRAS-mutant pancreatic tumours.

High-valency Anti-CD99 Antibodies Toward the Treatment of T Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that currently requires intensive chemotherapy. While childhood T-ALL is associated with high cure rates, adult T-ALL is not, and both are associated with significant short- and long-term morbidities. Thus, less toxic and effective strategies to treat T-ALL are needed. CD99 is overexpressed on T-ALL blasts at diagnosis and at relapse. Although targeting CD99 with cytotoxic antibodies has been proposed, the molecular features required for their activity are undefined. We identified human antibodies that selectively bound to the extracellular domain of human CD99, and the most potent clone, 10A1, shared an epitope with a previously described cytotoxic IgM antibody. We engineered clone 10A1 in bivalent, trivalent, tetravalent, and dodecavalent formats. Increasing the antibody valency beyond two had no effects on binding to T-ALL cells. In contrast, a valency of ≥3 was required for cytotoxicity, suggesting a mechanism of action in which an antibody clusters ≥3 CD99 molecules to induce cytotoxicity. We developed a human IgG-based tetravalent version of 10A1 that exhibited cytotoxic activity to T-ALL cells but not to healthy peripheral blood cells. The crystal structure of the 10A1 Fab in complex with a CD99 fragment revealed that the antibody primarily recognizes a proline-rich motif (PRM) of CD99 in a manner reminiscent of SH3-PRM interactions. This work further validates CD99 as a promising therapeutic target in T-ALL and defines a pathway toward the development of a selective therapy against T-ALL.

Mechanism of disease and therapeutic rescue of Dok7 congenital myasthenia.

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.

Impaired Humoral Immunity to SARS-CoV-2 Vaccination in Non-Hodgkin Lymphoma and CLL Patients.

Patients with hematologic malignancies are a high priority for SARS-CoV-2 vaccination, yet the benefit they will derive is uncertain. We investigated the humoral response to vaccination in 53 non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), or CLL patients. Peripheral blood was obtained 2 weeks after first vaccination and 6 weeks after second vaccination for antibody profiling using the multiplex bead-binding assay. Serum IgG, IgA, and IgM antibody levels to the spike specific receptor binding domain (RBD) were evaluated as a measure of response. Subsequently, antibody-positive serum were assayed for neutralization capacity against authentic SARS-CoV-2. Histology was 68% lymphoma and 32% CLL; groups were: patients receiving anti-CD20-based therapy (45%), monitored with disease (28%), receiving BTK inhibitors (19%), or chemotherapy (all HL) (8%). SARS-CoV-2 specific RBD IgG antibody response was decreased across all NHL and CLL groups: 25%, 73%, and 40%, respectively. Antibody IgG titers were significantly reduced (p < 0.001) for CD20 treated and targeted therapy patients, and (p = 0.003) for monitored patients. In 94% of patients evaluated after first and second vaccination, antibody titers did not significantly boost after second vaccination. Only 13% of CD20 treated and 13% of monitored patients generated neutralizing antibodies to SARS-CoV-2 with ICD50s 135 to 1767, and 445 and > 10240. This data has profound implications given the current guidance relaxing masking restrictions and for timing of vaccinations. Unless immunity is confirmed with laboratory testing, these patients should continue to mask, socially distance, and to avoid close contact with non-vaccinated individuals. STATEMENT OF TRANSLATIONAL RELEVANCE: Non Hodgkin lymphoma (NHL) and Chronic Lymphocytic leukemia (CLL) patients who are treated with anti-CD20 antibody therapy, BTK inhibitor therapy, or who are monitored with active disease, have decreased antibody response to SARS-CoV-2 vaccination and decreased antibody titers compared to healthy controls. Antibody titers do not boost following second vaccination, and very few patients generate neutralizing antibodies against SARS-CoV-2. This data is of particular importance, given the recent guidance from the CDC that vaccinated patients no longer need to be masked indoors as well as outdoors. Patients with NHL or CLL who fall into these categories should not consider their immunity from vaccination to be assured. If infected with SARS-CoV-2, they should be a high priority for monoclonal antibody directed therapy. Unless immune response to vaccination is confirmed with laboratory testing, they should continue to mask, socially distance, and to avoid close contact with non-vaccinated individuals.

Selective and noncovalent targeting of RAS mutants for inhibition and degradation.

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.

SHP2 inhibition diminishes KRASG12C cycling and promotes tumor microenvironment remodeling.

KRAS is the most frequently mutated human oncogene, and KRAS inhibition has been a longtime goal. Recently, inhibitors were developed that bind KRASG12C-GDP and react with Cys-12 (G12C-Is). Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that an SHP2 inhibitor (SHP2-I) increases KRAS-GDP occupancy, enhancing G12C-I efficacy. The SHP2-I abrogated RTK feedback signaling and adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). SHP2-I/G12C-I combination evoked favorable but tumor site-specific changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for PDAC regression and remodeling of the immune microenvironment but revealed direct inhibitory effects on tumor angiogenesis and vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional potential combination strategies.

Selective inhibition of STAT3 signaling using monobodies targeting the coiled-coil and N-terminal domains.

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms.

Allosteric modulation of a human protein kinase with monobodies.

Despite being the subject of intense effort and scrutiny, kinases have proven to be consistently challenging targets in inhibitor drug design. A key obstacle has been promiscuity and consequent adverse effects of drugs targeting the ATP binding site. Here we introduce an approach to controlling kinase activity by using monobodies that bind to the highly specific regulatory allosteric pocket of the oncoprotein Aurora A (AurA) kinase, thereby offering the potential for more specific kinase modulators. Strikingly, we identify a series of highly specific monobodies acting either as strong kinase inhibitors or activators via differential recognition of structural motifs in the allosteric pocket. X-ray crystal structures comparing AurA bound to activating vs inhibiting monobodies reveal the atomistic mechanism underlying allosteric modulation. The results reveal 3 major advantages of targeting allosteric vs orthosteric sites: extreme selectivity, ability to inhibit as well as activate, and avoidance of competing with ATP that is present at high concentrations in the cells. We envision that exploiting allosteric networks for inhibition or activation will provide a general, powerful pathway toward rational drug design.

Targeting the α4-α5 interface of RAS results in multiple levels of inhibition.

Generation of RAS-targeted therapeutics has long been considered a "holy grail" in cancer research. However, a lack of binding pockets on the surface of RAS and its picomolar affinity for guanine nucleotides have made isolation of inhibitors particularly challenging. We recently described a monobody, termed NS1, that blocks RAS signaling and oncogenic transformation. NS1 binds to the α4-ß6-α5 interface of H-RAS and K-RAS thus preventing RAS dimerization and nanoclustering, which in turn prevents RAS-stimulated dimerization and activation of RAF. Interestingly, NS1 reduces interaction of oncogenic K-RAS, but not H-RAS, with RAF and reduces K-RAS plasma membrane localization. Here, we show that these isoform specific effects of NS1 on RAS:RAF are due to the distinct hypervariable regions of RAS isoforms. NS1 inhibited wild type RAS function by reducing RAS GTP levels. These findings reveal that NS1 disrupts RAS signaling through a mechanism that is more complex than simply inhibiting RAS dimerization and nanoclustering.

Facile target validation in an animal model with intracellularly expressed monobodies.

Rapidly determining the biological effect of perturbing a site within a potential drug target could guide drug discovery efforts, but it remains challenging. Here, we describe a facile target validation approach that exploits monobodies, small synthetic binding proteins that can be fully functionally expressed in cells. We developed a potent and selective monobody to WDR5, a core component of the mixed lineage leukemia (MLL) methyltransferase complex. The monobody bound to the MLL interaction site of WDR5, the same binding site for small-molecule inhibitors whose efficacy has been demonstrated in cells but not in animals. As a genetically encoded reagent, the monobody inhibited proliferation of an MLL-AF9 cell line in vitro, suppressed its leukemogenesis and conferred a survival benefit in an in vivo mouse leukemia model. The capacity of this approach to readily bridge biochemical, structural, cellular characterization and tests in animal models may accelerate discovery and validation of druggable sites.

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT.

The maturation of RAS GTPases and approximately 200 other cellular CAAX proteins involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CAAX motif, proteolytic cleavage of the AAX residues and methylation of the exposed prenylcysteine residue at its terminal carboxylate. This final step is catalysed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryote-specific integral membrane enzyme that resides in the endoplasmic reticulum. ICMT is the only cellular enzyme that is known to methylate prenylcysteine substrates; methylation is important for the biological functions of these substrates, such as the membrane localization and subsequent activity of RAS, prelamin A and RAB. Inhibition of ICMT has potential for combating progeria and cancer. Here we present an X-ray structure of ICMT, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor, at 2.3 Å resolution. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for the cytosolic methyl donor, S-adenosyl-l-methionine, and for prenylcysteine substrates, which are associated with the endoplasmic reticulum membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localizations together in a membrane environment-a relatively uncharacterized but defining feature of many integral membrane enzymes.

Structural and functional dissection of the DH and PH domains of oncogenic Bcr-Abl tyrosine kinase.

The two isoforms of the Bcr-Abl tyrosine kinase, p210 and p190, are associated with different leukemias and have a dramatically different signaling network, despite similar kinase activity. To provide a molecular rationale for these observations, we study the Dbl-homology (DH) and Pleckstrin-homology (PH) domains of Bcr-Abl p210, which constitute the only structural differences to p190. Here we report high-resolution structures of the DH and PH domains and characterize conformations of the DH-PH unit in solution. Our structural and functional analyses show no evidence that the DH domain acts as a guanine nucleotide exchange factor, whereas the PH domain binds to various phosphatidylinositol-phosphates. PH-domain mutants alter subcellular localization and result in decreased interactions with p210-selective interaction partners. Hence, the PH domain, but not the DH domain, plays an important role in the formation of the differential p210 and p190 Bcr-Abl signaling networks.