SHP2-EGFR States in Dephosphorylation Can Inform Selective SHP2 Inhibitors, Dampening RasGAP Action.

SHP2 is a positive regulator of the EGFR-dependent Ras/MAPK pathway. It dephosphorylates a regulatory phosphorylation site in EGFR that serves as the binding site to RasGAP (RASA1 or p120RasGAP). RASA1 is activated by binding to the EGFR phosphate group. Active RASA1 deactivates Ras by hydrolyzing Ras-bound GTP to GDP. Thus, SHP2 dephosphorylation of EGFR effectively prevents RASA1-mediated deactivation of Ras, thereby stimulating proliferation. Despite knowledge of this vital regulation in cell life, mechanistic in-depth structural understanding of the involvement of SHP2, EGFR, and RASA1 in the Ras/MAPK pathway has largely remained elusive. Here we elucidate the interactions, the factors influencing EGFR's recruitment of RASA1, and SHP2's recognition of the substrate site in EGFR. We reveal that RASA1 specifically interacts with the DEpY992LIP motif in EGFR featuring a proline residue at the +3 position C-terminal to pY primarily through its nSH2 domain. This interaction is strengthened by the robust attraction of two acidic residues, E991 and D990, of EGFR to two basic residues in the BC-loop near the pY-binding pocket of RASA1's nSH2. In the stable precatalytic state of SHP2 with EGFR (DADEpY992LIPQ), the E-loop of SHP2's active site favors the interaction with the (-2)-position D990 and (-4)-position D988 N-terminal to pY992 in EGFR, while the pY-loop constrains the (+4)-position Q996 C-terminal to pY992. These specific interactions not only provide a structural basis for identifying negative regulatory sites in other RTKs but can inform selective, high-affinity active-site SHP2 inhibitors tailored for SHP2 mutants.

Slower CDK4 and faster CDK2 activation in the cell cycle.

Dysregulation of cyclin-dependent kinases (CDKs) impacts cell proliferation, driving cancer. Here, we ask why the cyclin-D/CDK4 complex governs cell cycle progression through the longer G1 phase, whereas cyclin-E/CDK2 regulates the shorter G1/S phase transition. We consider available experimental cellular and structural data including cyclin-E's high-level burst, sustained duration of elevated cyclin-D expression, and explicit solvent molecular dynamics simulations of the inactive monomeric and complexed states, to establish the conformational tendencies along the landscape of the distinct activation scenarios of cyclin-D/CDK4 and cyclin-E/CDK2 in the G1 phase and G1/S transition of the cell cycle, respectively. These lead us to propose slower activation of cyclin-D/CDK4 and rapid activation of cyclin-E/CDK2. We provide the mechanisms through which this occurs, offering innovative CDK4 drug design considerations. Our insightful mechanistic work addresses a compelling cell cycle regulation question and illuminates the distinct activation speeds between the G1 and the G1/S phases, which are crucial for function.

Disruption of Ca2+/calmodulin:KSR1 interaction lowers ERK activation.

KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca2+-binding protein calmodulin (CaM), thereby providing an intersection between KSR1-mediated and Ca2+ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca2+/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca2+/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca2+/CaM to a globular conformation of KSR1-CA3 via electrostatic interactions or (2) binding of collapsed Ca2+/CaM to α-helical KSR1-CA3 via hydrophobic interactions. Experimentally, site-directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca2+/CaM binds to the α-helical conformation of KSR1-CA3. Importantly, replacing KSR1-Phe355 with Asp reduces Ca2+/CaM binding by 76%. The KSR1-F355D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca2+/CaM binding promotes KSR1-mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca2+/CaM, identifies a KSR1 single-point mutant as a bioreagent to selectively study the crosstalk between Ca2+ and KSR1-mediated signaling.

Allosteric Activation of RhoA Complexed with p115-RhoGEF Deciphered by Conformational Dynamics.

The Ras homologue family member A (RhoA) is a member of the Rho family, a subgroup of the Ras superfamily. RhoA interacts with the 115 kDa guanine nucleotide exchange factor (p115-RhoGEF), which assists in activation and binding with downstream effectors. Here, we use molecular dynamics (MD) simulations and essential dynamics analysis of the inactive RhoA-GDP and active RhoA-GTP, when bound to p115-RhoGEF to decipher the mechanism of RhoA activation at the structural level. We observe that inactive RhoA-GDP maintains its position near the catalytic site on the Dbl homology (DH) domain of p115-RhoGEF through the interaction of its Switch I region with the DH domain. We further show that the active RhoA-GTP is engaged in more interactions with the p115-RhoGEF membrane-bound Pleckstrin homology (PH) domain as compared to RhoA-GDP. We hypothesize that the role of the interactions between the active RhoA-GTP and the PH domain is to help release it from the DH domain upon activation. Our results support this premise, and our simulations uncover the beginning of this process and provide structural details. They also point to allosteric communication pathways that take part in RhoA activation to promote and strengthen the interaction between the active RhoA-GTP and the PH domain. Allosteric regulation also occurs among other members of the Rho superfamily. Collectively, we suggest that in the activation process, the role of the RhoA-GTP interaction with the PH domain is to release RhoA-GTP from the DH domain after activation, making it available to downstream effectors.

The allosteric mechanism of mTOR activation can inform bitopic inhibitor optimization.

mTOR serine/threonine kinase is a cornerstone in the PI3K/AKT/mTOR pathway. Yet, the detailed mechanism of activation of its catalytic core is still unresolved, likely due to mTOR complexes' complexity. Its dysregulation was implicated in cancer and neurodevelopmental disorders. Using extensive molecular dynamics (MD) simulations and compiled published experimental data, we determine exactly how mTOR's inherent motifs can control the conformational changes in the kinase domain, thus kinase activity. We also chronicle the critical regulation by the unstructured negative regulator domain (NRD). When positioned inside the catalytic cleft (NRD IN state), mTOR tends to adopt a deep and closed catalytic cleft. This is primarily due to the direct interaction with the FKBP-rapamycin binding (FRB) domain which restricts it, preventing substrate access. Conversely, when outside the catalytic cleft (NRD OUT state), mTOR favors an open conformation, exposing the substrate-binding site on the FRB domain. We further show how an oncogenic mutation (L2427R) promotes shifting the mTOR ensemble toward the catalysis-favored state. Collectively, we extend mTOR's "active-site restriction" mechanism and clarify mutation action. In particular, our mechanism suggests that RMC-5552 (RMC-6272) bitopic inhibitors may benefit from adjustment of the (PEG8) linker length when targeting certain mTOR variants. In the cryo-EM mTOR/RMC-5552 structure, the distance between the allosteric and orthosteric inhibitors is ∼22.7 Å. With a closed catalytic cleft, this linker bridges the sites. However, in our activation mechanism, in the open cleft it expands to ∼24.7 Å, offering what we believe to be the first direct example of how discovering an activation mechanism can potentially increase the affinity of inhibitors targeting mutants.

Direct K-Ras Inhibitors to Treat Cancers: Progress, New Insights, and Approaches to Treat Resistance.

Here we discuss approaches to K-Ras inhibition and drug resistance scenarios. A breakthrough offered a covalent drug against K-RasG12C. Subsequent innovations harnessed same-allele drug combinations, as well as cotargeting K-RasG12C with a companion drug to upstream regulators or downstream kinases. However, primary, adaptive, and acquired resistance inevitably emerge. The preexisting mutation load can explain how even exceedingly rare mutations with unobservable effects can promote drug resistance, seeding growth of insensitive cell clones, and proliferation. Statistics confirm the expectation that most resistance-related mutations are in cis, pointing to the high probability of cooperative, same-allele effects. In addition to targeted Ras inhibitors and drug combinations, bifunctional molecules and innovative tri-complex inhibitors to target Ras mutants are also under development. Since the identities and potential contributions of preexisting and evolving mutations are unknown, selecting a pharmacologic combination is taxing. Collectively, our broad review outlines considerations and provides new insights into pharmacology and resistance.

Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI.

Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code," where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42, etc.). This work attempts to understand the molecular mechanism behind this specific phosphorylation-induced reduction in binding affinity. Using several microseconds long atomistic molecular dynamics simulations of the wild-type and phosphorylated states of the RhoA-RhoGDI complex, we propose a molecular-interaction-based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N-terminal region of RhoGDI that contribute most to the binding affinity. Molecular mechanics Poisson-Boltzmann surface area binding energy calculations show a significant weakening of interaction on phosphorylation at the RhoA-specific site of RhoGDI. In contrast, phosphorylation at a Rac1-specific site does not affect the overall binding affinity significantly, which confirms the presence of a phosphorylation code. RhoA-specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N-terminal region of RhoGDI, which manifests a reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long-range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.

SHP2 clinical phenotype, cancer, or RASopathies, can be predicted by mutant conformational propensities.

SHP2 phosphatase promotes full activation of the RTK-dependent Ras/MAPK pathway. Its mutations can drive cancer and RASopathies, a group of neurodevelopmental disorders (NDDs). Here we ask how same residue mutations in SHP2 can lead to both cancer and NDD phenotypes, and whether we can predict what the outcome will be. We collected and analyzed mutation data from the literature and cancer databases and performed molecular dynamics simulations of SHP2 mutants. We show that both cancer and Noonan syndrome (NS, a RASopathy) mutations favor catalysis-prone conformations. As to cancer versus RASopathies, we demonstrate that cancer mutations are more likely to accelerate SHP2 activation than the NS mutations at the same genomic loci, in line with NMR data for K-Ras4B more aggressive mutations. The compiled experimental data and dynamic features of SHP2 mutants lead us to propose that different from strong oncogenic mutations, SHP2 activation by NS mutations is less likely to induce a transition of the ensemble from the SHP2 inactive state to the active state. Strong signaling promotes cell proliferation, a hallmark of cancer. Weak, or moderate signals are associated with differentiation. In embryonic neural cells, dysregulated differentiation is connected to NDDs. Our innovative work offers structural guidelines for identifying and correlating mutations with clinical outcomes, and an explanation for why bearers of RASopathy mutations may have a higher probability of cancer. Finally, we propose a drug strategy against SHP2 variants-promoting cancer and RASopathies.

Neurodevelopmental disorders and cancer networks share pathways, but differ in mechanisms, signaling strength, and outcome.

Epidemiological studies suggest that individuals with neurodevelopmental disorders (NDDs) are more prone to develop certain types of cancer. Notably, however, the case statistics can be impacted by late discovery of cancer in individuals afflicted with NDDs, such as intellectual disorders, autism, and schizophrenia, which may bias the numbers. As to NDD-associated mutations, in most cases, they are germline while cancer mutations are sporadic, emerging during life. However, somatic mosaicism can spur NDDs, and cancer-related mutations can be germline. NDDs and cancer share proteins, pathways, and mutations. Here we ask (i) exactly which features they share, and (ii) how, despite their commonalities, they differ in clinical outcomes. To tackle these questions, we employed a statistical framework followed by network analysis. Our thorough exploration of the mutations, reconstructed disease-specific networks, pathways, and transcriptome levels and profiles of autism spectrum disorder (ASD) and cancers, point to signaling strength as the key factor: strong signaling promotes cell proliferation in cancer, and weaker (moderate) signaling impacts differentiation in ASD. Thus, we suggest that signaling strength, not activating mutations, can decide clinical outcome.

Spatial Mechano-Signaling Regulation of GTPases through Non-Degradative Ubiquitination.

Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear-stressed endothelial cells demonstrated that the non-degradative ubiquitination of several GTPases is regulated by mechano-signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2 is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non-degradative ubiquitination fine-tunes the function of GTPases by modifying their interacting network. Specifically, WWP2-mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1) toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress-induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial-specific Wwp2-knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non-degradative ubiquitination of GTPases in endothelial mechano-activation.

Protein conformational ensembles in function: roles and mechanisms.

The sequence-structure-function paradigm has dominated twentieth century molecular biology. The paradigm tacitly stipulated that for each sequence there exists a single, well-organized protein structure. Yet, to sustain cell life, function requires (i) that there be more than a single structure, (ii) that there be switching between the structures, and (iii) that the structures be incompletely organized. These fundamental tenets called for an updated sequence-conformational ensemble-function paradigm. The powerful energy landscape idea, which is the foundation of modernized molecular biology, imported the conformational ensemble framework from physics and chemistry. This framework embraces the recognition that proteins are dynamic and are always interconverting between conformational states with varying energies. The more stable the conformation the more populated it is. The changes in the populations of the states are required for cell life. As an example, in vivo, under physiological conditions, wild type kinases commonly populate their more stable "closed", inactive, conformations. However, there are minor populations of the "open", ligand-free states. Upon their stabilization, e.g., by high affinity interactions or mutations, their ensembles shift to occupy the active states. Here we discuss the role of conformational propensities in function. We provide multiple examples of diverse systems, including protein kinases, lipid kinases, and Ras GTPases, discuss diverse conformational mechanisms, and provide a broad outlook on protein ensembles in the cell. We propose that the number of molecules in the active state (inactive for repressors), determine protein function, and that the dynamic, relative conformational propensities, rather than the rigid structures, are the hallmark of cell life.

Cancer and Autism: How PTEN Mutations Degrade Function at the Membrane and Isoform Expression in the Human Brain.

Mutations causing loss of PTEN lipid phosphatase activity can promote cancer, benign tumors (PHTS), and neurodevelopmental disorders (NDDs). Exactly how they preferentially trigger distinct phenotypic outcomes has been puzzling. Here, we demonstrate that PTEN mutations differentially allosterically bias P loop dynamics and its connection to the catalytic site, affecting catalytic activity. NDD-related mutations are likely to sample conformations of the functional wild-type state, while sampled conformations for the strong, cancer-related driver mutation hotspots favor catalysis-primed conformations, suggesting that NDD mutations are likely to be weaker, and our large-scale simulations show why. Prenatal PTEN isoform expression data suggest exons 5 and 7, which harbor NDD mutations, as cancer-risk carriers. Since cancer requires more than a single mutation, our conformational and genomic analysis helps discover how same protein mutations can foster different clinical manifestations, articulates a role for co-occurring background latent driver mutations, and uncovers relationships of splicing isoform expression to life expectancy.

Cell cycle progression mechanisms: slower cyclin-D/CDK4 activation and faster cyclin-E/CDK2.

Dysregulation of cyclin-dependent kinases (CDKs) impacts cell proliferation, driving cancer. Here, we ask why the cyclin-D/CDK4 complex governs cell cycle progression through the longer G1 phase, whereas cyclin-E/CDK2 regulates the short G1/S phase transition. We consider the experimentally established high-level bursting of cyclin-E, and sustained duration of elevated cyclin-D expression in the cell, available experimental cellular and structural data, and comprehensive explicit solvent molecular dynamics simulations to provide the mechanistic foundation of the distinct activation scenarios of cyclin-D/CDK4 and cyclin-E/CDK2 in the G1 phase and G1/S transition of the cell cycle, respectively. These lead us to propose slower activation of cyclin-D/CDK4 and rapid activation of cyclin-E/CDK2. Importantly, we determine the mechanisms through which this occurs, offering innovative CDK4 drug design considerations. Our insightful mechanistic work addresses the compelling cell cycle regulation question and illuminates the distinct activation speeds in the G1 versus G1/S phases, which are crucial for cell function.

Cell phenotypes can be predicted from propensities of protein conformations.

Proteins exist as dynamic conformational ensembles. Here we suggest that the propensities of the conformations can be predictors of cell function. The conformational states that the molecules preferentially visit can be viewed as phenotypic determinants, and their mutations work by altering the relative propensities, thus the cell phenotype. Our examples include (i) inactive state variants harboring cancer driver mutations that present active state-like conformational features, as in K-Ras4BG12V compared to other K-Ras4BG12X mutations; (ii) mutants of the same protein presenting vastly different phenotypic and clinical profiles: cancer and neurodevelopmental disorders; (iii) alterations in the occupancies of the conformational (sub)states influencing enzyme reactivity. Thus, protein conformational propensities can determine cell fate. They can also suggest the allosteric drugs efficiency.

Neurodevelopmental disorders, like cancer, are connected to impaired chromatin remodelers, PI3K/mTOR, and PAK1-regulated MAPK.

Neurodevelopmental disorders (NDDs) and cancer share proteins, pathways, and mutations. Their clinical symptoms are different. However, individuals with NDDs have higher probabilities of eventually developing cancer. Here, we review the literature and ask how the shared features can lead to different medical conditions and why having an NDD first can increase the chances of malignancy. To explore these vital questions, we focus on dysregulated PI3K/mTOR, a major brain cell growth pathway in differentiation, and MAPK, a critical pathway in proliferation, a hallmark of cancer. Differentiation is governed by chromatin organization, making aberrant chromatin remodelers highly likely agents in NDDs. Dysregulated chromatin organization and accessibility influence the lineage of specific cell brain types at specific embryonic development stages. PAK1, with pivotal roles in brain development and in cancer, also regulates MAPK. We review, clarify, and connect dysregulated pathways with dysregulated proliferation and differentiation in cancer and NDDs and highlight PAK1 role in brain development and MAPK regulation. Exactly how PAK1 activation controls brain development, and why specific chromatin remodeler components, e.g., BAF170 encoded by SMARCC2 in autism, await clarification.

AlphaFold, allosteric, and orthosteric drug discovery: Ways forward.

Drug discovery is arguably a highly challenging and significant interdisciplinary aim. The stunning success of the artificial intelligence-powered AlphaFold, whose latest version is buttressed by an innovative machine-learning approach that integrates physical and biological knowledge about protein structures, raised drug discovery hopes that unsurprisingly, have not come to bear. Even though accurate, the models are rigid, including the drug pockets. AlphaFold's mixed performance poses the question of how its power can be harnessed in drug discovery. Here we discuss possible ways of going forward wielding its strengths, while bearing in mind what AlphaFold can and cannot do. For kinases and receptors, an input enriched in active (ON) state models can better AlphaFold's chance of rational drug design success.

Small GTPase Ran: Depicting the nucleotide-specific conformational landscape of the functionally important C-terminus.

The small GTPase Ran is the main regulator of the nucleo-cytoplasmic import and export through the nuclear pore complex. It functions as a molecular switch cycling between the GDP-bound inactive and GTP-bound active state. It consists of a globular (G) domain and a C-terminal region, which is bound to the G-domain in the inactive, GDP-bound states. Crystal structures of the GTP-bound active form complexed with Ran binding proteins (RanBP) show that the C-terminus undergoes a large conformational change, embracing Ran binding domains (RanBD). Whereas in the crystal structures of macromolecular complexes not containing RanBDs the structure of the C-terminal segment remains unresolved, indicating its large conformational flexibility. This movement could not have been followed either by experimental or simulation methods. Here, starting from the crystal structure of Ran in both GDP- and GTP-bound forms we show how rigid the C-terminal region in the inactive structure is during molecular dynamics (MD) simulations. Furthermore, we show how MD simulations of the active form are incapable of mapping the open conformations of the C-terminus. By using the MDeNM (Molecular Dynamics with excited Normal Modes) method, we were able to widely map the conformational surface of the C-terminus of Ran in the active GTP-bound form, which allows us to envisage how it can embrace RanBDs.

How PTEN mutations degrade function at the membrane and life expectancy of carriers of mutations in the human brain.

PTEN dysfunction, caused by loss of lipid phosphatase activity or deletion, promotes pathologies, cancer, benign tumors, and neurodevelopmental disorders (NDDs). Despite efforts, exactly how the mutations trigger distinct phenotypic outcomes, cancer or NDD, has been puzzling. It has also been unclear how to distinguish between mutations harbored by isoforms, are they cancer or NDDs-related. Here we address both. We demonstrate that PTEN mutations differentially allosterically bias P-loop dynamics and its connection to the catalytic site, affecting catalytic activity. NDD-related mutations are likely to sample conformations present in the wild-type, while sampled conformations sheltering cancer-related hotspots favor catalysis-prone conformations, suggesting that NDD mutations are weaker. Analysis of isoform expression data indicates that if the transcript has NDD-related mutations, alone or in combination with cancer hotspots, there is high prenatal expression. If no mutations within the measured days, low expression levels. Cancer mutations promote stronger signaling and cell proliferation; NDDs' are weaker, influencing brain cell differentiation. Further, exon 5 is impacted by NDD or non-NDD mutations, while exon 7 is exclusively impacted by NDD mutations. Our comprehensive conformational and genomic analysis helps discover how same allele mutations can foster different clinical manifestations and uncovers correlations of splicing isoform expression to life expectancy.

Strategy toward Kinase-Selective Drug Discovery.

Kinase drug selectivity is the ground challenge in cancer research. Due to the structurally similar kinase drug pockets, off-target inhibitor toxicity has been a major cause for clinical trial failures. The pockets are similar but not identical. Here, we describe a transformation invariant protocol to identify distinct geometric features in the drug pocket that can distinguish one kinase from all others. We integrate available experimental structures with the artificial intelligence-based structural kinome, performing a kinome-wide structural bioinformatic analysis to establish the structural principles of kinase drug selectivity. We generate the structural landscape from the experimental kinase-ligand complexes and propose a binary network that encapsulates the information. The results show that all kinases contain binary units that are shared by less than seven other kinases in the kinome. 331 kinases contain unique binary units that may distinguish them from all others. The structural features encoded by these binary units in the network represent the inhibitor-accessible geometric space that may capture the kinome-wide selectivity. Our proposed binary network with the unsupervised clustering can serve as a general structural bioinformatic protocol for extracting the distinguishing structural features for any protein from their families. We apply the binary network to epidermal growth factor receptor tyrosine kinase inhibitor selectivity by targeting the gate area and the AKT1 serine/threonine kinase selectivity by binding to the αC-helix region and the allosteric pocket. Finally, we develop the cross-platform software, KDS (Kinase Drug Selectivity), for customized visualization and analysis of the binary networks in the human kinome (https://github.com/CBIIT/KDS).

Higher-order interactions of Bcr-Abl can broaden chronic myeloid leukemia (CML) drug repertoire.

Bcr-Abl, a nonreceptor tyrosine kinase, is associated with leukemias, especially chronic myeloid leukemia (CML). Deletion of Abl's N-terminal region, to which myristoyl is linked, renders the Bcr-Abl fusion oncoprotein constitutively active. The substitution of Abl's N-terminal region by Bcr enables Bcr-Abl oligomerization. Oligomerization is critical: it promotes clustering on the membrane, which is essential for potent MAPK signaling and cell proliferation. Here we decipher the Bcr-Abl specific, step-by-step oligomerization process, identify a specific packing surface, determine exactly how the process is structured and identify its key elements. Bcr's coiled coil (CC) domain at the N-terminal controls Bcr-Abl oligomerization. Crystallography validated oligomerization via Bcr-Abl dimerization between two Bcr CC domains, with tetramerization via tight packing between two binary assemblies. However, the structural principles guiding Bcr CC domain oligomerization are unknown, hindering mechanistic understanding and drugs exploiting it. Using molecular dynamics (MD) simulations, we determine that the binary complex of the Bcr CC domain serves as a basic unit in the quaternary complex providing a specific surface for dimer-dimer packing and higher-order oligomerization. We discover that the small α1-helix is the key. In the binary assembly, the helix forms interchain aromatic dimeric packing, and in the quaternary assembly, it contributes to the specific dimer-dimer packing. Our mechanism is supported by the experimental literature. It offers the key elements controlling this process which can expand the drug discovery strategy, including by Bcr CC-derived peptides, and candidate residues for small covalent drugs, toward quenching oligomerization, supplementing competitive and allosteric tyrosine kinase inhibitors.