Mutations in RAS and PI3Kα are major drivers of human cancer. Their interaction plays a crucial role in activating PI3Kα and amplifying the PI3K-AKT-mTOR pathway. Disrupting RAS-PI3Kα interaction enhances survival in lung and skin cancer models and reduces tumor growth and angiogenesis, although the structural details of this interaction remain unclear. Here, we present structures of KRAS, RRAS2, and MRAS bound to the catalytic subunit (p110α) of PI3Kα, elucidating the interaction interfaces and local conformational changes upon complex formation. Structural and mutational analyses highlighted key residues in RAS and PI3Kα impacting binding affinity and revealed isoform-specific differences at the interaction interface in RAS and PI3K isoforms, providing a rationale for their differential affinities. Notably, in the RAS-p110α complex structures, RAS interaction with p110α is limited to the RAS-binding domain and does not involve the kinase domain. This study underscores the pivotal role of the RAS-PI3Kα interaction in PI3Kα activation and provides a blueprint for designing PI3Kα isoform-specific inhibitors to disrupt this interaction.

SARS-CoV-2 infections led to a worldwide pandemic in 2020. As of 2024, therapeutics against SARS-CoV-2 have continued to be desirable. NSP14 is a dual-function methyltransferase (MTase) and exonuclease (ExoN) with key roles in SARS-CoV-2 genome propagation and host immune system evasion. In this work, we developed high-throughput screening (HTS) assays for NSP14 MTase and ExoN activities. We screened both activities against a collection of 40,664 compounds. A total of 1677 initial hit compounds were identified, cherrypicked, counterscreened for assay interference, and screened for off-target selectivity. We identified 396 and 174 high-quality hits against the MTase and ExoN activities, respectively. Along with inhibitors for individual activities, we identified dual-activity inhibitors, including a novel inhibitor that is not competitive with any substrate and interacts with a putative allosteric binding site. This study represents the largest published screen of SARS-CoV-2 NSP14 MTase and ExoN activities to date and culminates in a pipeline for the NSP14 drug discovery.

KRAS , the most frequently mutated oncogene in human cancer, produces two isoforms, KRAS4a and KRAS4b, through alternative splicing. These isoforms differ in exon 4, which encodes the final 15 residues of the G-domain and hypervariable regions (HVRs), vital for trafficking and membrane localization. While KRAS4b has been extensively studied, KRAS4a has been largely overlooked. Our multidisciplinary study compared the structural and functional characteristics of KRAS4a and KRAS4b, revealing distinct structural properties and thermal stability. Position 151 influences KRAS4a’s thermal stability, while position 153 affects binding to RAF1 CRD protein. Nuclear magnetic resonance analysis identified localized structural differences near sequence variations and provided a solution-state conformational ensemble. Notably, KRAS4a exhibits substantial transcript abundance in bile ducts, liver, and stomach, with transcript levels approaching KRAS4b in the colon and rectum. Functional disparities were observed in full-length KRAS variants, highlighting the impact of HVR variations on interaction with trafficking proteins and downstream effectors like RAF and PI3K within cells.

Tobacco-etch-virus (TEV) protease is the workhorse of many laboratories in which protein expression is the linchpin of downstream experiments. TEV protease is remarkable in its sequence specificity as the cleavage sequence rarely appears in higher organisms and its ability to cleave fusion tag proteins from proteins of interest. Herein we report work done on large-scale production of TEV protease using different promotors, media, fusion tags, and expression platforms. During our work we detected post-translational modification (gluconoylation and phosphogluconoylation) of TEV protease and the subsequent effects this has on the purity of the protein. Subsequently we made our pgl plus bacteria that negates these modifications and their effects. We also introduce a GFP-based assay for measurement of activity and ultimately a new set of protocols for producing 400-500 mg/L TEV protease.

Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110α/p85α and a 3-fold increase in p110α. We also used these protocols to produce comparable constructs of the β and δ isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases.

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.

Abstract The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli.

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.

We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform. This method allows us to comprehensively evaluate compound specificity, clearly detect nonspecific binding events, and determine the order and frequency with which they occur. We provide two proof-of-concept examples of our method employing publicly available compounds, along with detailed protocols for sample preparation, top-down MS data acquisition, targeted proteoform MS2 fragmentation, and analysis of the resulting data. Our results demonstrate the concentration dependence of KRAS4B-compound engagement and highlight the ability of top-down MS to directly map compound binding location(s) without disrupting the KRAS4B primary structure. Our hope is that this novel method may help accelerate the identification of new successful targeted inhibitors for KRAS4B and other RAS isoforms.

The RAS family of GTPases (HRAS, KRAS, NRAS) continue to present formidable challenges to the design of compounds that successfully and specifically target oncogenic RAS mutant populations in cancer. Although recent advances have led to the successful development of inhibitors specific to KRAS G12C (such as Sotorasib [Amgen] and Adagrasib [Mirati Therapeutics]), ongoing efforts seek to develop additional targeted inhibitors for oncogenic mutants of KRAS. Development of new KRAS targeted inhibitors requires the understanding of how a mutation impacts the accessibility of key residues within the protein, along with how each candidate compound interacts with populations of mutant and wild-type proteins. Proteomic analyses, typically comprising peptide-based methods to confirm compound localization or quantify binding kinetics, represent a key validation step for each phase of the inhibitor development process. However, these methods may not always provide a clear picture of how effectively the compound binds to the protein, how specific the compound is to the target residue, and how concentration or incubation time may impact these factors. To address this, we have developed a novel assay to evaluate target engagement in vitro employing top-down proteomics, which entails the analysis of intact protein molecules and their modified forms (proteoforms). By analyzing intact labeled or compound-treated recombinant KRAS4B proteins, we can directly visualize and precisely characterize each species within a sample. This method allows us to easily detect nonspecific binding, identify targeting of incorrect residues, and characterize unanticipated compound reaction products within a short LC-MS run, all facilitated by targeted MS2 fragmentation. Moreover, we have developed complementary methods for RAS proteoform quantitation, allowing for the evaluation of compound engagement to all proteoforms with greater accuracy than traditional peptide-based methods. We first performed a proof-of-concept experiment using a panel of KRAS4B G12D_C118S proteins with selected residues modified to cysteine to evaluate reactivity to maleimide-biotin over time. We then applied our optimized top-down assay to determine the engagement of targeted covalent inhibitors Sotorasib and Adagrasib with KRAS WT, G12C, or G13C in both the active (GppNHp) and inactive (GDPβS) states. Finally, we selected three compounds from a large-scale screen against a UCSF Small Molecule Discovery Center compound library and evaluated each for specificity to KRAS4B G13C over KRAS4B WT and KRAS4B G12C. Our results show the concentration dependence of target engagement and directly map compound binding location(s) without disrupting the protein’s primary structure. Moreover, our ability to target discrete compound additions for top-down MS2 fragmentation provides context and order of preferential labeling of KRAS4B. Our hope is that this methodology may help accelerate the identification of new successful targeted inhibitors for KRAS and other RAS isoforms by the greater RAS community. Citation Format: Robert A. D'Ippolito, Caroline J. DeHart, Dana Rabara, Maria Abreu Blanco, Emily Alberico, Nitya Ramakrishnan, Stephanie Widmeyer, Simon Messing, David Turner, Michelle Arkin, Anna Maciag, Andrew Stephen, Dominic Esposito, Frank McCormick, Dwight V. Nissley. A top-down proteomic assay to evaluate KRAS4B-compound engagement [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A018.

RIT1 is a RAS guanosine triphosphatase (GTPase) that regulates different aspects of signal transduction and is mutated in lung cancer, leukemia, and in the germline of individuals with Noonan syndrome. Pathogenic RIT1 proteins promote mitogen-activated protein kinase (MAPK) hyperactivation; however, this mechanism remains poorly understood. Here, we show that RAF kinases are direct effectors of membrane-bound mutant RIT1 necessary for MAPK activation. We identify critical residues in RIT1 that facilitate interaction with membrane lipids and show that these are necessary for association with RAF kinases and MAPK activation. Although mutant RIT1 binds to RAF kinases directly, it fails to activate MAPK signaling in the absence of classical RAS proteins. Consistent with aberrant RAF/MAPK activation as a driver of disease, we show that pathway inhibition alleviates cardiac hypertrophy in a mouse model of RIT1 mutant Noonan syndrome. These data shed light on the function of pathogenic RIT1 and identify avenues for therapeutic intervention.

KRAS is the most frequently mutated RAS protein in cancer patients, and it is estimated that about 20% of the cancer patients in the United States carried mutant RAS proteins. To accelerate therapeutic development, structures and dynamics of RAS proteins had been extensively studied by various biophysical techniques for decades. Although 31P NMR studies revealed population equilibrium of the two major states in the active GMPPNP-bound form, more complex conformational dynamics in RAS proteins and oncogenic mutants subtly modulate the interactions with their downstream effectors. We established a set of customized NMR relaxation dispersion techniques to efficiently and systematically examine the ms-μs conformational dynamics of RAS proteins. This method allowed us to observe varying synchronized motions that connect the effector and allosteric lobes in KRAS. We demonstrated the role of conformational dynamics of KRAS in controlling its interaction with the Ras-binding domain of the downstream effector RAF1, the first kinase in the MAPK pathway. This allows one to explain, as well as to predict, the altered binding affinities of various KRAS mutants, which was neither previously reported nor apparent from the structural perspective.

SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of the SHOC2–MRAS–PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS, and PP1C form a stable ternary complex in which all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex forms only when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation and how mutations found in Noonan syndrome enhance complex formation, and reveal new avenues for therapeutic interventions. The high-resolution crystal structure of the SHOC2–MRAS–PP1C complex provides insights into the complex assembly, RAF dephosphorylation, MRAS selectivity versus canonical RAS isoforms, and the impact of Noonan syndrome mutations on complex formation.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates in the United States and elsewhere. To address this, we analyzed seropositivity in 9089 adults in the United States who had not been diagnosed previously with COVID-19. Individuals with characteristics that reflected the U.S. population ( n = 27,716) were selected by quota sampling from 462,949 volunteers. Enrolled participants ( n = 11,382) provided medical, geographic, demographic, and socioeconomic information and dried blood samples. Survey questions coincident with the Behavioral Risk Factor Surveillance System survey, a large probability-based national survey, were used to adjust for selection bias. Most blood samples (88.7%) were collected between 10 May and 31 July 2020 and were processed using ELISA to measure seropositivity (IgG and IgM antibodies against SARS-CoV-2 spike protein and the spike protein receptor binding domain). The overall weighted undiagnosed seropositivity estimate was 4.6% (95% CI, 2.6 to 6.5%), with race, age, sex, ethnicity, and urban/rural subgroup estimates ranging from 1.1% to 14.2%. The highest seropositivity estimates were in African American participants; younger, female, and Hispanic participants; and residents of urban centers. These data indicate that there were 4.8 undiagnosed SARS-CoV-2 infections for every diagnosed case of COVID-19, and an estimated 16.8 million infections were undiagnosed by mid-July 2020 in the United States.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection.

The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is key to avoiding medically costly diagnostic errors, as well as to assuring properly informed public health decisions. Here, we present an optimized ELISA-based serology protocol, from antigen production to data analyses, that helps define thresholds for IgG and IgM seropositivity with high specificities. Validation of this protocol is performed using traditionally collected serum as well as dried blood on mail-in blood sampling kits. Archival (pre-2019) samples are used as negative controls, and convalescent, PCR-diagnosed COVID-19 patient samples serve as positive controls. Using this protocol, minimal cross-reactivity is observed for the spike proteins of MERS, SARS1, OC43 and HKU1 viruses, and no cross reactivity is observed with anti-influenza A H1N1 HAI. Our protocol may thus help provide standardized, population-based data on the extent of SARS-CoV-2 seropositivity, immunity and infection.

Recombinant mammalian proteins are routinely produced in E. coli and thus lack post-translational modifications. KRAS4b is processed at both the N- and C-terminus, resulting in an acetylation of the N-terminus (at Thr, after aminopeptidase removal of the original N-term Met) and farnesylation/carboxymethylation of the C-terminal Cys (after proteolytic cleavage of the original C-terminal three amino acids, Val-Iso-Met). Processing of KRAS enables it to associate with the plasma membrane and fulfill its function in cell signaling. We describe here the production of recombinant KRAS4b from our modified baculovirus/insect cell expression system that accurately incorporates these in vivo modifications to allow experiments that anchor KRAS4b to membrane mimetics (e.g., nanodiscs and liposomes).

Significance RAS proteins control many aspects of cellular biology in response to extracellular stimuli. These essential nodes of signal transduction interact with effector proteins that contain conserved RAS-binding domains. Mutations in RAS proteins are a common cause of cancer; hence, understanding the function of their downstream effectors is important for the development of novel therapies. In this study, we characterize the interaction between RAS and the effector protein Sin1, a critical component of the mTORC2, through biochemical and structural approaches. We discover that Sin1 contains an atypical RAS-binding domain and assess the role of this interaction in cells and mice. Moreover, we find that this interaction is dispensable for mTORC2 activity and assembly, suggesting an alternative function for Sin1 in cells.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates. To address this, we analyzed seropositivity in US adults who have not previously been diagnosed with COVID-19. Individuals with characteristics that reflect the US population (n = 11,382) and who had not previously been diagnosed with COVID-19 were selected by quota sampling from 241,424 volunteers (ClinicalTrials.gov NCT04334954). Enrolled participants provided medical, geographic, demographic, and socioeconomic information and 9,028 blood samples. The majority (88.7%) of samples were collected between May 10th and July 31st, 2020. Samples were analyzed via ELISA for anti-Spike and anti-RBD antibodies. Estimation of seroprevalence was performed by using a weighted analysis to reflect the US population. We detected an undiagnosed seropositivity rate of 4.6% (95% CI: 2.6 - 6.5%). There was distinct regional variability, with heightened seropositivity in locations of early outbreaks. Subgroup analysis demonstrated that the highest estimated undiagnosed seropositivity within groups was detected in younger participants (ages 18-45, 5.9%), females (5.5%), Black/African American (14.2%), Hispanic (6.1%), and Urban residents (5.3%), and lower undiagnosed seropositivity in those with chronic diseases. During the first wave of infection over the spring/summer of 2020 an estimate of 4.6% of adults had a prior undiagnosed SARS-CoV-2 infection. These data indicate that there were 4.8 (95% CI: 2.8-6.8) undiagnosed cases for every diagnosed case of COVID-19 during this same time period in the United States, and an estimated 16.8 million undiagnosed cases by mid-July 2020.