Meredith L. Jenkins

Conformational disruption of PI3Kδ regulation by immunodeficiency mutations in PIK3CD and PIK3R1

Significance Activated PI3K Delta Syndrome (APDS) is a primary immunodeficiency disease caused by activating mutations in phosphoinositide 3-kinases (PI3Kδ). Activating mutations in either the p110δ catalytic or the p85α regulatory subunit of PI3Kδ result in APDS. Mutations in p85α leading to APDS are surprising, as other p85α-activating mutations are oncogenic when bound to the PI3Kα isoform. Using hydrogen–deuterium exchange mass spectrometry, we determined the molecular mechanisms by which APDS mutations in p110δ or p85α activate PI3Kδ and reveal why the p85α APDS2 mutant primarily activates PI3Kδ. All APDS mutants are potently inhibited by the PI3Kδ-specific inhibitor idelalisib. Together, the biophysical and biochemical data reveal insights into PI3Kδ regulation and provide a possible therapeutic strategy for treating patients with APDS. Activated PI3K Delta Syndrome (APDS) is a primary immunodeficiency disease caused by activating mutations in either the leukocyte-restricted p110δ catalytic (PIK3CD) subunit or the ubiquitously expressed p85α regulatory (PIK3R1) subunit of class IA phosphoinositide 3-kinases (PI3Ks). There are two classes of APDS: APDS1 that arises from p110δ mutations that are analogous to oncogenic mutations found in the broadly expressed p110α subunit and APDS2 that occurs from a splice mutation resulting in p85α with a central deletion (Δ434–475). As p85 regulatory subunits associate with and inhibit all class IA catalytic subunits, APDS2 mutations are expected to similarly activate p110α, β, and δ, yet APDS2 largely phenocopies APDS1 without dramatic effects outside the immune system. We have examined the molecular mechanism of activation of both classes of APDS mutations using a combination of biochemical assays and hydrogen–deuterium exchange mass spectrometry. Intriguingly, we find that an APDS2 mutation in p85α leads to substantial basal activation of p110δ (>300-fold) and disrupts inhibitory interactions from the nSH2, iSH2, and cSH2 domains of p85, whereas p110α is only minimally basally activated (∼2-fold) when associated with mutated p85α. APDS1 mutations in p110δ (N334K, E525K, E1021K) mimic the activation mechanisms previously discovered for oncogenic mutations in p110α. All APDS mutations were potently inhibited by the Food and Drug Administration-approved p110δ inhibitor idelalisib. Our results define the molecular basis of how PIK3CD and PIK3R1 mutations result in APDS and reveal a potential path to treatment for all APDS patients.

Molecular mechanism of activation of class IA phosphoinositide 3-kinases (PI3Ks) by membrane-localized HRas

Class IA PI3Ks are involved in the generation of the key lipid signaling molecule phosphatidylinositol 3,4,5-trisphosphate (PIP3), and inappropriate activation of this pathway is implicated in a multitude of human diseases, including cancer, inflammation, and primary immunodeficiencies. Class IA PI3Ks are activated downstream of the Ras superfamily of GTPases, and Ras–PI3K interaction plays a key role in promoting tumor formation and maintenance in Ras-driven tumors. Investigating the detailed molecular events in the Ras–PI3K interaction has been challenging because it occurs on a membrane surface. Here, using maleimide-functionalized lipid vesicles, we successfully generated membrane-resident HRas and evaluated its effect on PI3K signaling in lipid kinase assays and through analysis with hydrogen–deuterium exchange MS. We screened all class IA PI3K isoforms and found that HRas activates both p110α and p110δ isoforms but does not activate p110β. The p110α and p110δ activation by Ras was synergistic with activation by a soluble phosphopeptide derived from receptor tyrosine kinases. Hydrogen–deuterium exchange MS revealed that membrane-resident HRas, but not soluble HRas, enhances conformational changes associated with membrane binding by increasing membrane recruitment of both p110α and p110δ. Together, these results afford detailed molecular insight into the Ras–PI3K signaling complex, provide a framework for screening Ras inhibitors, and shed light on the isoform specificity of Ras–PI3K interactions in a native membrane context.

Using Hydrogen–Deuterium Exchange Mass Spectrometry to Examine Protein–Membrane Interactions

Many fundamental cellular processes are controlled via assembly of a network of proteins at membrane surfaces. The proper recruitment of proteins to membranes can be controlled by a wide variety of mechanisms, including protein lipidation, protein-protein interactions, posttranslational modifications, and binding to specific lipid species present in membranes. There are, however, only a limited number of analytical techniques that can study the assembly of protein-membrane complexes at the molecular level. A relatively new addition to the set of techniques available to study these protein-membrane systems is the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS experiments measure protein conformational dynamics in their native state, based on the rate of exchange of amide hydrogens with solvent. This review discusses the use of HDX-MS as a tool to identify the interfaces of proteins with membranes and membrane-associated proteins, as well as define conformational changes elicited by membrane recruitment. Specific examples will focus on the use of HDX-MS to examine how large macromolecular protein complexes are recruited and activated on membranes, and how both posttranslational modifications and cancer-linked oncogenic mutations affect these processes.

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches.

Molecular basis for recognition of the cancer glycobiomarker LacdiNAc (GalNAc(β1-4)GlcNAc) by Wisteria floribunda agglutinin

Aberrant glycosylation and the overexpression of specific carbohydrate epitopes is a hallmark of many cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy and diagnostics. Wisteria floribunda agglutinin (WFA) is a legume lectin that recognizes terminal N-acetylgalactosaminides with high affinity. WFA preferentially binds the disaccharide LacdiNAc (β-d-GalNAc-[1→4]-d-GlcNAc), which is associated with tumor malignancy in leukemia, prostate, pancreatic, ovarian, and liver cancers and has shown promise in cancer glycobiomarker detection. The mechanism of specificity for WFA recognition of LacdiNAc is not fully understood. To address this problem, we have determined affinities and structure of WFA in complex with GalNAc and LacdiNAc. Affinities toward Gal, GalNAc, and LacdiNAc were measured via surface plasmon resonance, yielding KD values of 4.67 × 10−4 m, 9.24 × 10−5 m, and 5.45 × 10−6 m, respectively. Structures of WFA in complex with LacdiNAc and GalNAc have been determined to 1.80–2.32 Å resolution. These high resolution structures revealed a hydrophobic groove complementary to the GalNAc and, to a minor extent, to the back-face of the GlcNAc sugar ring. Remarkably, the contribution of this small hydrophobic surface significantly increases the observed affinity for LacdiNAc over GalNAc. Tandem MS sequencing confirmed the presence of two isolectin forms in commercially available WFA differing only in the identities of two amino acids. Finally, the WFA carbohydrate binding site is similar to a homologous lectin isolated from Vatairea macrocarpa in complex with GalNAc, which, unlike WFA, binds not only αGalNAc but also terminal Ser/Thr O-linked αGalNAc (Tn antigen).

An overview of hydrogen deuterium exchange mass spectrometry (HDX-MS) in drug discovery

ABSTRACT Introduction: Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful methodology to study protein dynamics, protein folding, protein-protein interactions, and protein small molecule interactions. The development of novel methodologies and technical advancements in mass spectrometers has greatly expanded the accessibility and acceptance of this technique within both academia and industry. Areas covered: This review examines the theoretical basis of how amide exchange occurs, how different mass spectrometer approaches can be used for HDX-MS experiments, as well as the use of HDX-MS in drug development, specifically focusing on how HDX-MS is used to characterize bio-therapeutics, and its use in examining protein-protein and protein small molecule interactions. Expert opinion: HDX-MS has been widely accepted within the pharmaceutical industry for the characterization of bio-therapeutics as well as in the mapping of antibody drug epitopes. However, there is room for this technique to be more widely used in the drug discovery process. This is particularly true in the use of HDX-MS as a complement to other high-resolution structural approaches, as well as in the development of small molecule therapeutics that can target both active-site and allosteric binding sites.

Expanding the Scope of Electrophiles Capable of Targeting K-Ras Oncogenes

There is growing interest in reversible and irreversible covalent inhibitors that target noncatalytic amino acids in target proteins. With a goal of targeting oncogenic K-Ras variants (e.g., G12D) by expanding the types of amino acids that can be targeted by covalent inhibitors, we survey a set of electrophiles for their ability to label carboxylates. We functionalized an optimized ligand for the K-Ras switch II pocket with a set of electrophiles previously reported to react with carboxylates and characterized the ability of these compounds to react with model nucleophiles and oncogenic K-Ras proteins. Here, we report that aziridines and stabilized diazo groups preferentially react with free carboxylates over thiols. Although we did not identify a warhead that potently labels K-Ras G12D, we were able to study the interactions of many electrophiles with K-Ras, as most of the electrophiles rapidly label K-Ras G12C. We characterized the resulting complexes by crystallography, hydrogen/deuterium exchange, and differential scanning fluorimetry. Our results both demonstrate the ability of a noncatalytic cysteine to react with a diverse set of electrophiles and emphasize the importance of proper spatial arrangements between a covalent inhibitor and its intended nucleophile. We hope that these results can expand the range of electrophiles and nucleophiles of use in covalent protein modulation.

An intrinsic lipid-binding interface controls sphingosine kinase 1 function

Sphingosine kinase 1 (SK1) is required for production of sphingosine-1-phosphate (S1P) and thereby regulates many cellular processes, including cellular growth, immune cell trafficking, and inflammation. To produce S1P, SK1 must access sphingosine directly from membranes. However, the molecular mechanisms underlying SK1’s direct membrane interactions remain unclear. We used hydrogen/deuterium exchange MS to study interactions of SK1 with membrane vesicles. Using the CRISPR/Cas9 technique to generate HCT116 cells lacking SK1, we explored the effects of membrane interface disruption and the function of the SK1 interaction site. Disrupting the interface resulted in reduced membrane association and decreased cellular SK1 activity. Moreover, SK1-dependent signaling, including cell invasion and endocytosis, was abolished upon mutation of the membrane-binding interface. Of note, we identified a positively charged motif on SK1 that is responsible for electrostatic interactions with membranes. Furthermore, we demonstrated that SK1 uses a single contiguous interface, consisting of an electrostatic site and a hydrophobic site, to interact with membrane-associated anionic phospholipids. Altogether, these results define a composite domain in SK1 that regulates its intrinsic ability to bind membranes and indicate that this binding is critical for proper SK1 function. This work will allow for a new line of thinking for targeting SK1 in disease.

Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex

Apicomplexan parasites such as Toxoplasma gondii rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA–light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775–818) using a combination of hydrogen–deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm Kd measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a Kd of 12 nm). Using the full-length MyoA motor (residues 1–831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction.

Recognition of protein-linked glycans as a determinant of peptidase activity.

Significance Protein glycosylation is one of the most abundant and important posttranslational modifications where the protein-linked glycans can impart specific physiochemical properties to the glycoprotein and/or the glycans themselves can mediate particular biological functions. The degradation of glycosylated proteins in normal or pathogenic processes, therefore, is an important biological process. This study reveals the molecular basis of how peptidases can use the O-glycans present on glycoproteins as a critical determinant of peptidase activity and, in doing so, provides unique insight into how peptidases may directly use posttranslational modifications present on their substrates to influence recognition and peptide bond cleavage. The vast majority of proteins are posttranslationally altered, with the addition of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate posttranslational modifications, including glycosylation, has not been addressed. Through biochemical analyses and X-ray crystallographic structures we show that to hydrolyze their substrates, three structurally related metallopeptidases require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The three peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular understanding of glycoprotein degradation.