The influence of adnectin binding on the extracellular domain of epidermal growth factor receptor.
Iacob RE, Chen G, Ahn J, Houel S, Wei H, Mo J, Tao L, Cohen D, Xie D, Lin Z, Morin PE, Doyle ML,
Tymiak AA, Engen JR.

J Am Soc Mass Spectrom. 2014. Dec;25(12):2093-2102.

The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity.   More recently, smaller protein derived from the tenth type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies.  The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS).  The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography. 
Pubmed: 25223306

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A conserved isoleucine maintains the inactive state of Bruton's tyrosine kinase.
Boyken SE, Chopra N, Xie Q, Joseph RE, Wales TE, Fulton DB, Engen JR, Jernigan RL, Andreotti AH.
J Mol Biol. 2014. Oct 23;426(21):3656-3669.

Despite high homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamic simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state.  The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest this isoleucine side chain in the Tec family kinases acts as a ‘wedge’ that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain.
Pubmed: 25193673

Differential sensitivity of Src-family kinases to activation by SH3 domain displacement.
Moroco JA, Craigo JK, Iacob RE, Wales TE, Engen JR, Smithgall TE.
PLoS One. 2014. Aug 21;9(8):e105629.


Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.

Pubmed: 25144189

Electron transfer control in soluble methane monooxygenase.
Wang W, Iacob RE, Luoh RP, Engen JR, Lippard SJ.
J Am Chem Soc. 2014. Jul 9;136(27):9754-62.


The hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases (BMM) requires the interplay of three or four protein components. How component protein interactions control catalysis, however, is not well understood. In particular, the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic. Here we determined the reductase binding site on the hydroxylase of a BMM enzyme, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). We present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase, previously determined to be the regulatory protein binding site as well. The latter thus inhibits reductase binding to the hydroxylase and, consequently, intermolecular electron transfer from the reductase to the hydroxylase diiron active site. The binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer, and other BMM enzymes are likely to adopt the same mechanism.  

Pubmed: 24937475

GroEL/ES chaperonin modulates the mechanism and accelerates the rate of TIM-barrel domain folding.
Georgescauld F, Popova K, Gupta AJ, Bracher A, Engen JR, Hayer-Hartl M, Hartl FU.
Cell. 2014. May 8;157(4):922-934.


The GroEL/ES chaperonin system functions as a protein folding cage. Many obligate substrates of GroEL share the (βα)8 TIM-barrel fold, but how the chaperonin promotes folding of these proteins is not known. Here, we analyzed the folding of DapA at peptide resolution using hydrogen/deuterium exchange and mass spectrometry. During spontaneous folding, all elements of the DapA TIM barrel acquire structure simultaneously in a process associated with a long search time. In contrast, GroEL/ES accelerates folding more than 30-fold by catalyzing segmental structure formation in the TIM barrel. Segmental structure formation is also observed during the fast spontaneous folding of a structural homolog of DapA from a bacterium that lacks GroEL/ES. Thus, chaperonin independence correlates with folding properties otherwise enforced by protein confinement in the GroEL/ES cage. We suggest that folding catalysis by GroEL/ES is required by a set of proteins to reach native state at a biologically relevant timescale, avoiding aggregation or degradation.   

Pubmed: 24813614

Conformational analysis of processivity clamps in solution demonstrates that tertiary structure does not correlate with protein dynamics.
Fang J, Nevin P, Kairys V, Venclovas C, Engen JR, Beuning PJ.
Structure. 2014. Apr 8;22(4):572-581.


The relationship between protein sequence, structure, and dynamics has been elusive. We report one of the first comprehensive analyses using an in-solution experimental approach to study how the conservation of tertiary structure correlates with protein dynamics. Hydrogen exchange measurements of eight processivity clamp proteins from different species revealed that, despite highly similar three-dimensional structures, clamp proteins display a wide range of dynamic behavior. Differences were apparent both for structurally similar domains within proteins and for corresponding domains of different proteins. Several of the clamps contained regions that underwent local unfolding with different half-lives. We also observed a conserved pattern of alternating dynamics of the α-helices lining the inner pore of the clamps as well as a correlation between dynamics and the number of salt bridges in these α-helices. Our observations reveal that tertiary structure and dynamics are not directly correlated and that primary structure plays an important role in dynamics.   

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Pubmed: 24613485

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A conformational investigation of propeptide binding to the integral membrane protein γ-glutamyl carboxylase using nanodisc hydrogen exchange mass spectrometry.
Parker CH, Morgan CR, Rand KD, Engen JR, Jorgenson JW, Stafford DW.
Biochemistry 2014. Mar 11;53(9):1511-1520.


Gamma (γ)-glutamyl carboxylase (GGCX) is an integral membrane protein responsible for the post-translational catalytic conversion of select glutamic acid (Glu) residues to γ-carboxy glutamic acid (Gla) in vitamin K-dependent (VKD) proteins. Understanding the mechanism of carboxylation and the role of GGCX in the vitamin K cycle is of biological interest in the development of therapeutics for blood coagulation disorders. Historically, biophysical investigations and structural characterizations of GGCX have been limited due to complexities involving the availability of an appropriate model membrane system. In previous work, a hydrogen exchange mass spectrometry (HX MS) platform was developed to study the structural configuration of GGCX in a near-native nanodisc phospholipid environment. Here we have applied the nanodisc-HX MS approach to characterize specific domains of GGCX that exhibit structural rearrangements upon binding the high-affinity consensus propeptide (pCon; AVFLSREQANQVLQRRRR). pCon binding was shown to be specific for monomeric GGCX-nanodiscs and promoted enhanced structural stability to the nanodisc-integrated complex while maintaining catalytic activity in the presence of carboxylation co-substrates. Noteworthy modifications in HX of GGCX were prominently observed in GGCX peptides 491–507 and 395–401 upon pCon association, consistent with regions previously identified as sites for propeptide and glutamate binding. Several additional protein regions exhibited minor gains in solvent protection upon propeptide incorporation, providing evidence for a structural reorientation of the GGCX complex in association with VKD carboxylation. The results herein demonstrate that nanodisc-HX MS can be utilized to study molecular interactions of membrane-bound enzymes in the absence of a complete three-dimensional structure and to map dynamic rearrangements induced upon ligand binding.

Pubmed: 24512177

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Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor.
Lim SM, Westover KD, Ficarro S, Harrison RA, Choi HG, Pacold ME, Carrasco M, Hunter J, Kim ND, Xie T, Sim T, Janne PA, Meyerson M, Marto JA, Engen JR, Gray NS.
Angew. Chem. Int. Ed. 2014. Jan 3;53(1):199-204.


We report the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggest that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. These first-in-class covalent K-Ras inhibitors demonstrate that irreversible targeting of the K-Ras guanine-nucleotide binding site is potentially a viable therapeutic strategy for inhibition of Ras signaling.   

Pubmed: 24259466

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Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: Methodology and applications.
Wei H, Mo J, Tao L, Russell RJ, Tymiak AA, Chen G, Iacob RE, Engen JR.

Drug Discov Today. 2014. Jan 1;19(1):95-102.

The higher order structure of protein therapeutics can be interrogated with hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS is now a widely used tool in the structural characterization of protein therapeutics. In this review, HDX-MS-based workflows designed for protein therapeutic discovery and development processes are presented, focusing on the specific applications of epitope mapping for protein and/or drug interactions and biopharmaceutical comparability studies. Future trends in the application of HDX-MS in protein therapeutics characterization are also described.
Pubmed: 23928097

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Hydrogen exchange mass spectrometry measures stapled peptide conformational dynamics and predicts pharmacokinetic properties.
Shi X, Wales TE, Elkin C, Kawahata N, Engen JR, Annis DA.
Anal. Chem. 2013. Dec 3;85(23):11185-11188.


Peptide drugs have traditionally suffered from poor pharmacokinetic properties due to their conformational flexibility and the interaction of proteases with backbone amide bonds.  “Stapled Peptides” are cyclized using an all-hydrocarbon cross-linking strategy to reinforce their α-helical conformation, yielding improved protease resistance and drug-like properties.  Here we demonstrate that Hydrogen Exchange-Mass Spectrometry (HX-MS) effectively probes the conformational dynamics of Stapled Peptides derived from the survivin-borealin protein-protein interface and predicts their susceptibility to proteolytic degradation.  In Stapled Peptides, amide exchange was reduced by over five orders-of-magnitude versus the native peptide sequence depending on staple placement.  Furthermore, deuteration kinetics correlated directly with rates of proteolysis to reveal the optimal staple placement for improved drug properties.   

Pubmed: 24215480

Analysis of overlapped and noisy Hydrogen/Deuterium exchange data.
Guttman M, Weis DD, Engen JR, Lee KK.
J Am Soc Mass Spectrom. 2013. Dec 1;24(12):1906-1912

Noisy and overlapped mass spectrometry data hinders the sequence coverage that can be obtained from Hydrogen Deuterium exchange analysis, and places a limit on the complexity of the samples that can be studied by this technique. Advances in instrumentation have expanded these limits, but as the complexity of the biological samples under investigation increases, these problems are reencountered. Here we describe the use of binomial distribution fitting with asymmetric linear squares regression for calculating the accurate deuterium content for mass envelopes of low signal or that contain significant overlap. The approach is demonstrated with a test data set of HIV Env gp140 wherein inclusion of the new analysis regime resulted in obtaining exchange data for 42 additional peptides, improving the sequence coverage by 10%. At the same time, the precision of deuterium uptake measurements was improved for nearly every peptide examined. The improved processing algorithms also provide an efficient method for deconvolution of bimodal mass envelopes and EX1 kinetic signatures. All these functions and visualization tools have been implemented in the new version of the freely available software, HX-Express v2
Pubmed: 24018862

Investigating monoclonal antibody aggregation using a combination of H/DX-MS and other biophysical measurements.
Iacob RE, Bou-Assaf GM, Makowski L, Engen JR, Berkowitz SA, Houde D.
J. Pharm. Sci. 2013. Dec 1;102(12):4315-4329.


To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified.  In this work the higher-order structure and biophysical properties of three different monoclonal antibody (mAb) monomers was compared to their simplest aggregated form, i.e., dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry (H/DX-MS) and other biophysical measurements was used to make the comparison.  The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen deuterium exchange compared to its dimer form.  In this case, differences observed were located in specific functional regions of the CH2 domain and the hinge region between CH1 and CH2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.   

Pubmed: 24136070

Conformational transition of membrane-associated terminally-acylated HIV-1 Nef.

Akgun B, Satija S, Nanda H, Pirrone GF, Shi X, Engen JR, Kent MS.
Structure. 2013. Oct 8;21(10):1822-1833.

Many proteins are post-translationally modified by acylation targeting them to lipid membranes. While methods such as X-ray crystallography and NMR are available to determine the structure of folded proteins in solution, the precise position of folded domains relative to a membrane remains largely unknown. We used neutron and X-ray reflection methods to measure the displacement of the core domain of HIV Nef from lipid membranes upon insertion of the N-terminal myristate group. Nef is one of several HIV-1 accessory proteins and an essential factor in AIDS progression.  Upon insertion of the myristate and residues from the N-terminal arm, Nef transitions from a closed to open conformation that positions the core domain 70 Å from the lipid headgroups.  This work rules out speculation of Jia et al (Nat. Struct. Mol. Biol., 2012) that the Nef core remains closely associated with the membrane to optimize interactions with the cytoplasmic domain of MHC-1.

Pubmed: 24035710

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Insights into Notch3 activation and inhibition mediated by antibodies directed against its negative regulatory region.
Tiyanont K, Wales TE, Siebel CW, Engen JR, Blacklow SC.
J Mol Biol. 2013. Sep 9;425(17):3192-3204.


Notch receptors are single-pass transmembrane proteins that regulate development and tissue homeostasis in all metazoan organisms. Prior to ligand-induced signaling, Notch receptors adopt a proteolytic-resistant conformation maintained by a critical interdomain interface within a negative regulatory region (NRR), which sits immediately external to the plasma membrane. Signaling is initiated when ligand binding induces exposure of the proteolytic cleavage site, termed S2, within the NRR.  Here, we use hydrogen exchange in conjunction with mass spectrometry (HX-MS) to study the dynamics of the human Notch3 NRR in four distinct biochemical states: in its unmodified quiescent form, in a proteolytically “on” state induced by EDTA, and in complex with either agonist or inhibitory antibodies. Induction of the “on” state by either EDTA or the agonist monoclonal antibody leads to accelerated deuteration in the region of the S2 cleavage site, reflecting an increase in S2 dynamics. In contrast, complexation of the Notch3 NRR with an inhibitory antibody retards deteuration not only across its discontinuous binding epitope, but also around the S2 site, stabilizing the NRR in its “off” state. Together with previous work investigating the dynamics of the Notch1 NRR, these studies show that key features of autoinhibition and activation are shared among different Notch receptors, and provide additional insights into mechanisms of Notch activation and inhibition by modulatory antibodies. 

Pubmed: 23747483

Activation loop dynamics determine the different catalytic efficiencies of B cell- and T cell-specific Tec kinases.
Joseph RE, Kleino I, Wales TE, Xie Q, Fulton DB, Engen JR, Berg LJ, Andreotti AH.
Sci. Signal. 2013. Aug 27;6(290):ra76.

Itk (interleukin-2–inducible T cell kinase) and Btk (Bruton’s tyrosine kinase) are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of 6 of the 619 amino acid residues of Itk with the corresponding residues of Btk (and vice versa) was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties, depending on the cellular context. We suggest that the weaker catalytic activities of T cell–specific kinases serve to regulate cellular activation and prevent aberrant immune responses. 
Pubmed: 23982207

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Active-site inhibitors modulate the dynamic properties of human monoacylglycerol lipase: A hydrogen exchange mass spectrometry study.
Karageorgos I, Wales TE, Janero DR, Zvonok N, Vemuri VK, Engen JR, Makriyannis A.
Biochemistry. 2013. Jul 23;52(29):5016-5026.


Human monoacylglycerol lipase (hMGL) regulates endocannabinoid signaling primarily by deactivating the lipid messenger 2-arachidonoylglycerol.  Agents that carbamylate hMGL’s catalytic Ser122 constitute the leading class of therapeutically promising hMGL inhibitors. We have applied peptide-level hydrogen/deuterium exchange mass spectrometry to characterize hMGL’s conformational responses to two potent carbamate inhibitors, AM6580 (acutely irreversible) and AM6701 (slowly reversible).  A dynamic, solvent-exposed lid domain is characteristic of hMGL’s solution conformation.  Both hMGL inhibitors restricted backbone enzyme motility in the active-site region and increased substrate binding-pocket solvent exposure. Covalent reaction of AM6580 with hMGL generates a bulkier carbamylated Ser122 residue as compared to the more discrete Ser122 modification by AM6701, a difference reflected in AM6580’s more pronounced effect upon hMGL conformation.  We demonstrate that structurally distinct carbamate hMGL inhibitors generate particular conformational ensembles characterized by region-specific hMGL dynamics.  By demonstrating the distinctive influences of two hMGL inhibitors on enzyme conformation, this study furthers our understanding at the molecular level of the dynamic features of hMGL interaction with small-molecule ligands. 

Pubmed: 23795559

Non-canonical role of the PDZ4 domain of the adaptor protein PDZK1 in the regulation of the hepatic HDL receptor SR-BI.
Tsukamoto K, Wales TE, Daniels K, Pal R, Sheng R, Cho W, Stafford W, Engen JR, Krieger M, Kocher O.
J. Biol. Chem. 2013. Jul 5;288(27):19845-19860.


The four PDZ (PDZ1-PDZ4) domain-containing-adaptor protein PDZK1 controls the expression, localization and function of the HDL receptor SR-BI in hepatocytes in vivo via a PDZ4-dependent mechanism involving the binding of SR-BI’s cytoplasmic carboxy terminus to the canonical peptide binding sites of the PDZ1 or PDZ3 domains (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (DPDZ2 or DPDZ3), domain replacement (PDZ2→1) or target peptide binding-negative (PDZ4[G389P]) mutants of PDZK1, we found that neither PDZ2 nor PDZ3, nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell-surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifu-gation and hydrogen deuterium exchange mass spectrometry suggested that the requirement for PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, Surface Plasmon Resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1’s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and non-canonical (peptide binding independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.  

Pubmed: 23720744

Membrane phospholipid bilayer as a determinant of monoacylglycerol lipase kinetic profile and conformational repertoire.
Nasr ML, Shi X, Bowman AL, Johnson M, Zvonok N, Janero DR, Vemuri VK, Wales TE, Engen JR, Makriyannis A.
Protein Sci. 2013. Jun 1;22(6):774-787.


The membrane-associated serine hydrolase, monoacylglycerol lipase (MGL), is a well-recognized therapeutic target that regulates endocannabinoid signaling. Crystallographic studies, while providing structural information about static MGL states, offer no direct experimental insight into the impact of MGL's membrane association upon its structure-function landscape. We report application of phospholipid bilayer nanodiscs as biomembrane models with which to evaluate the effect of a membrane system on the catalytic properties and conformational dynamics of human MGL (hMGL). Anionic and charge-neutral phospholipid bilayer nanodiscs enhanced hMGL's kinetic properties [apparent maximum velocity (Vmax), substrate affinity (Km)]. Hydrogen-exchange mass spectrometry (HX MS) was used as a conformational analysis method to profile experimentally the extent of hMGL-nanodisc interaction and its impact upon hMGL structure. We provide evidence that significant regions of hMGL lid-domain helix α4 and neighboring helix α6 interact with the nanodisc phospholipid bilayer, anchoring hMGL in a more open conformation to facilitate ligand access to the enzyme's substrate-binding channel. Covalent modification of membrane-associated hMGL by the irreversible carbamate inhibitor, AM6580, shielded the active-site region, but did not increase solvent exposure of the lid domain, suggesting that the inactive, carbamylated enzyme remains intact and membrane associated. Molecular dynamics simulations generated conformational models congruent with the open, membrane-associated topology of active and inhibited, covalently-modified hMGL. Our data indicate that hMGL interaction with a phospholipid membrane bilayer induces regional changes in the enzyme's conformation that favor its recruiting lipophilic substrate/inhibitor from membrane stores to the active site via the lid, resulting in enhanced hMGL catalytic activity and substrate affinity. 

Pubmed: 23553709

Considerations in the analysis of hydrogen exchange mass spectrometry data.
Wales TE, Eggertson MJ, Engen JR.
Methods Mol Biol. 2013. 1007:263-288. 


A major component of a hydrogen exchange mass spectrometry experiment is the analysis of protein and peptide mass spectra to yield information about deuterium incorporation.  The processing of data that are produced includes the identification of each peptic peptide to create a master table/array of peptide sequence, retention time and retention time range, mass range and undeuterated mass.  The amount of deuterium incorporated into each of the peptides in this array must then be determined.  Various software platforms have been developed in order to perform this specific type of data analysis.  We describe the fundamental parameters to be considered at each step along the way and how data processing, either by an individual or by software, must approach the analysis. 

Pubmed: 23666730

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Accessing the reproducibility and specificity of pepsin and other aspartic proteases.
Ahn J, Cao M-C, Yu YQ, Engen JR.
Biochim Biophys Acta. 2013. Jun 1;1834(6):1222-1229.

The aspartic protease pepsin is less specific than other endoproteinases.  Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions.  We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion.  The collection of reproducible peptides was identified from >30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5-6 replicate digestions.  We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin.  Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine.  Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data.
Pubmed: 23063535

Conformational analysis of recombinant monoclonal antibodies with Hydrogen / Deuterium exchange mass spectrometry.
Houde D, Engen JR.
Methods Mol Biol. 2013. 988:269-289. 


Understanding the conformation of antibodies, especially those of therapeutic value, is of great interest.  Many of the current analytical methods used to probe protein conformation face issues in the analysis of antibodies, either due to the nature of the antibody itself or the limitations of the method.  One method that has recently been utilized for conformational analysis of antibodies is hydrogen/deuterium exchange mass spectrometry (H/DX MS).  H/DX MS can be used to probe the conformation and dynamics of proteins in solution, requires small sample quantities, is compatible with many buffer systems, and provides peptide-level resolution.  The application of H/DX MS to immunoglobulin gamma 1 (IgG1) recombinant monoclonal antibodies can provide information about IgG1 conformation, dynamics, and changes to conformation as a result of protein modification(s), changes in storage conditions, purification procedures, formulation, and many other parameters.  In this article we provide a comprehensive HD/X MS protocol for the analysis of an antibody. 

Pubmed: 23475726

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Enhanced SH3:linker interaction overcomes Abl kinase activation by gatekeeper and myristic acid binding pocket mutations & increases sensitivity to small molecule inhibitors.
Panjarian S, Iacob RE, Chen S, Wales TE, Engen JR, Smithgall TE.
J. Biol. Chem. 2013. Mar 1;288(9):6116-6129.

Multi-domain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their non-catalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3:linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3:linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with CML to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multi-domain kinase systems that uses multiple small molecules to exploit natural mechanisms of kinase control.

Pubmed: 23303187

Partial cooperative unfolding in proteins as observed by hydrogen exchange mass spectrometry.
Engen JR, Wales TE, Chen S, Marzluff EM, Hassell KM, Weis DD, Smithgall TE.
Int. Rev. Phys. Chem. 2013. Jan-Mar;23(1):96-127.

Many proteins do not exist in a single rigid conformation.  Protein motions, or dynamics, exist and in many cases are important for protein function.  The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion.  Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days.  In this review we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples.  Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of inter- and intra-molecular interactions in the global control of kinase function.  Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signaling proteins.  HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both classification of and greater understanding of the prevalence of protein motion. 

Pubmed: 23682200

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Structure and dynamic regulation of Abl kinases.
Panjarian S, Iacob RE, Chen S, Engen JR, Smithgall TE.
J. Biol. Chem. 2013. Feb 22;288(8):5443-5450. Invited minireview.

The c-Abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as regulation of the actin cytoskeleton.  Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia (CML).  Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in CML and revolutionized the field of targeted cancer therapy. In this mini-review, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity.

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Pubmed: 23316053

The use of Hydrogen/Deuterium exchange mass spectrometry in epitope mapping.
Ahn J, Engen JR.
Chimica Oggi / Chemistry Today. 2013. Jan/Feb;31(1):25-28. 

Discovery and development of effective biologic drugs such as antibodies are challenging tasks.  Understanding the antibody/antigen interaction and determining the epitope are important components of therapeutic antibody design and eventual commercialization.  In recent years, various techniques have been described for epitope mapping, each with its own strengths and weaknesses.  Here we highlight the mass spectrometry (MS) based approach of hydrogen/deuterium exchange (HDX) and describe its utility in epitope mapping.  While there are many advantages to HDX MS for epitope mapping, there are also challenges.  After a description of how a typical HDX MS epitope mapping experiment can be performed, some cautionary notes about the technique are presented.

Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors.
Yu W, Chory EJ, Wernimont AK, Tempel W, Scopton A, Federation A, Marineau JJ, Qi J, Barsyte-Lovejoy D, Yi J, Marcellus R, Iacob RE, Engen JR, Griffin C,Aman A, Wienholds E, Li F, Pineda J, Estiu G, Shatseva T, Hajian T, Al-Awar R, Dick JE, Vedadi M, Brown PJ, Arrowsmith CH, Bradner JE, Schapira M.
Nat Commun. 2012. Dec 18;3:1288.

Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound to EPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

Pubmed: 23250418

Pepsin immobilized on high-strength hybrid particles for continuous flow online digestion at 10,000 psi.
Ahn J, Jung MC, Wyndham K, Yu YQ, Engen JR.
Anal. Chem. 2012. Aug 21;84(16):7256-62.

Pepsin was immobilized on ethyl-bridged hybrid (BEH) particles and digestion performance was evaluated in a completely online format, with the specific intent of using the particles for hydrogen deuterium exchange mass spectrometry (HDX MS) experiments.  Because the BEH particles are mechanically strong, they could withstand prolonged, continuous high-pressure at 10,000 psi.  Online digestion was performed under isobaric conditions with continuous solvent flow, in contrast to other approaches where the pressure or flow is cycled.  As expected, digestion efficiency at 10,000 psi was increased and reproducibly produced more peptic peptides versus digestion at 1,000 psi.  Prototype columns made with the BEH pepsin particles exhibited robust performance and deuterium back-exchange was similar to that of other immobilized pepsin particles.  These particles can be easily incorporated in existing HDX MS workflows to provide more peptide coverage in experiments where fast, efficient, and reproducible online pepsin digestion is desired.
Pubmed: 22856522

Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars.
Berkowitz SA, Engen JR, Mazzeo JR, Jones GB.
Nat Rev Drug Discov. 2012. Jul 1;11(7): 527-540.


Biologics such as monoclonal antibodies are much more complex than small-molecule drugs, which raises challenging questions for the development and regulatory evaluation of follow‑on versions of such biopharmaceutical products (also known as biosimilars) and their clinical use once patent protection for the pioneering biologic has expired. With the recent introduction of regulatory pathways for follow‑on versions of complex biologics, the role of analytical technologies in comparing biosimilars with the reference product is attracting substantial interest in establishing the development requirements for biosimilars. Here, we discuss the current state of the art in analytical technologies to assess three characteristics of protein biopharmaceuticals that regulatory authorities have identified as being important in development strategies for biosimilars: post-translational modifications, three-dimensional structures and protein aggregation.  

Pubmed: 22743980

Hydrogen exchange mass spectrometry: Are we out of the quicksand?
Iacob RE, Engen JR.
J Am Soc Mass Spectrom. 2012. Jun;23(6):1003-1010. Invited 'Critical Insight'.

Although the use of hydrogen exchange (HX) mass spectrometry (MS) to study proteins and protein conformation is now over 20 years old, the perception lingers that it still has “issues”.  Is this method, in fact, still in the quicksand with many remaining obstacles to overcome?  We do not think so.  This critical insight addresses the “issues” and explores several broad questions including: have the limitations of HX MS been surmounted and has HX MS achieved “indispensable” status in the pantheon of protein structural analysis tools. 
Pubmed: 22476891

Endocannabinoid enzyme engineering: Soluble human thio-monoacylglycerol lipase (sol-S-hMGL).
Karageorgos I, Zvonok N, Janero DR, Vemuri VK, Shukla V, Wales TE, Engen JR, Makriyannis A.
ACS Chem Neurosci. 2012. May 16;3(5):393-399.


In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu169;Leu176)-to-Ser(Ser169;Ser176) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser122 with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser122 residue as compared to that of Cys122 in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys122 residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser122, and the mechanisms underlying hMGL inhibition by different classes of small molecules. 

Pubmed: 22860208

HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck.
Pene-Dumitrescu T, Shu ST, Wales TE, Alvarado JJ, Shi H, Narute PS, Moroco JA, Yeh JI, Engen JR,
Smithgall TE.

BMC Chem Biol. 2012. Apr;12(1):1.

Background: Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association.
Results: To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency.
Conclusions: These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.

Pubmed: 22420777

Using Hydrogen/Deuterium exchange mass spectrometry to study conformational changes in granulocyte colony stimulating factor upon PEGylation.
Wei H, Ahn J, Yu YQ, Tymiak AA, Engen JR, Chen G.
J Am Soc Mass Spectrom. 2012. Mar;23(3):498-504.

PEGylation is the covalent attachment of polyethylene glycol to proteins, and it can be used to alter immunogenicity, circulating half life and other properties of therapeutic proteins. To determine the impact of PEGylation on protein conformation, we applied hydrogen/deuterium exchange mass spectrometry (HDX MS) to analyze Granulocyte Colony Stimulating Factor (G-CSF) upon PEGylation as a model system. The combined use of HDX automation technology and data analysis software allowed reproducible and robust measurements of the deuterium incorporation levels for peptic peptides of both PEGylated and non-PEGylated G-CSF. The results indicated that significant differences in deuterium incorporation were induced by PEGylation of G-CSF, although the overall changes observed were quite small. PEGylation did not result in gross conformational rearrangement of G-CSF. The data complexity often encountered in HDX MS measurements was greatly reduced though a data processing and presentation format designed to facilitate the comparison process. This study demonstrates the practical utility of HDX MS for comparability studies, process monitoring and protein therapeutic characterization in the biopharmaceutical industry.
Pubmed: 22227798

Conformational locking upon cooperative assembly of Notch transcription complexes.
Choi SH, Wales TE, Nam Y, O'Donovan D, Sliz P, Engen JR, Blacklow SC.
Structure. 2012. Feb 8;20(2):340-349.


The Notch intracellular domain (NICD) forms a transcriptional activation complex with the DNA-binding factor CSL and a transcriptional co-activator of the Mastermind family (MAML). The "RAM" region of NICD recruits Notch to CSL, facilitating the binding of MAML at the interface between the ankyrin (ANK) repeat domain of NICD and CSL. Here, we report the X-ray structure of a human MAML1/RAM/ANK/CSL/DNA complex, and probe changes in component dynamics upon stepwise assembly of a MAML1/NICD/CSL complex using HX-MS.  Association of CSL with NICD exerts remarkably little effect on the exchange kinetics of the ANK domain, whereas MAML1 binding greatly retards the exchange kinetics of ANK repeats 2-3. These exchange patterns identify critical features contributing to the cooperative assembly of Notch transcription complexes (NTCs), highlight the importance of MAML recruitment in rigidifying the ANK domain and stabilizing its interface with CSL, and rationalize the requirement for MAML1 in driving cooperative dimerization of NTCs on paired site DNA.

Preview by R. Kovall

Pubmed: 22325781