Click here to read
Conformational insight into multi-protein signaling assemblies by hydrogen-deuterium exchange mass spectrometry.
Harrison RA, Engen JR.
Curr Opin Struct Biol. 2016. Dec;41:187-193.

Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) can provide information about proteins that can be challenging to obtain by other means. Structure/function relationships, binding interactions, and the effects of modification have all been measured with HDX MS for a diverse and growing array of signaling proteins and multiprotein signaling complexes. As a result of hardware and software improvements, receptors and complexes involved in cellular signaling – including those associated with membranes – can now be studied. The growing body of HDX MS studies of signaling complexes at membranes is particularly exciting. Recent examples are presented to illustrate what can be learned about signaling proteins with this technique.
Pubmed: 27552080

Click here to read
Structural dynamics in Ras and related proteins upon nucleotide switching.
Harrison RA, Lu J, Carrasco M, Hunter J, Manandhar A, Gondi S, Westover KD, Engen JR.
J Mol Biol. 2016. Nov 20;428(23):4723-4735.

Structural dynamics of Ras proteins contribute to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras targeting strategies, experimental methods to measure Ras dynamics are required. Here we demonstrate the utility of hydrogen-deuterium exchange mass spectrometry to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main chain movement. Hydrogen-deuterium exchange provides a means of evaluating Ras protein dynamics which may be useful for understanding mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics.
Pubmed: 27751724

Icon for American Chemical Society
c-Abl tyrosine kinase adopts multiple active conformational states in solution.
Badger J, Grover P, Panjarian SB, Engen JR, Smithgall TE, Makowski L.

Biochemistry. 2016. Jun 14;55(23):3251-3260.                                                 ACS Editors' Choice - 06/03/2016

Protein-tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here we present X-ray scattering solution structures of multi-domain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory Ncap, SH3 and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity.  
Pubmed: 27166638

Allosteric inhibition of antiapoptotic MCL-1.
Lee S, Wales TE, Escudero S, Cohen DT, Luccarelli J, Gallagher CG, Cohen NA, Huhn AJ, Bird GH,
Engen JR, Walensky LD.

Nat Struct Mol Biol. 2016. Jun 7;23(6):600-607.


MCL-1 is an antiapoptotic BCL-2 family protein that has emerged as a major pathogenic factor in human cancer. Like BCL-2, MCL-1 bears a surface groove whose function is to sequester the BH3 killer domains of proapoptotic BCL-2 family members, a mechanism harnessed by cancer cells to establish formidable apoptotic blockades. Although drugging the BH3-binding groove has been achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small-molecule covalent modification of C286 at a new interaction site distant from the BH3-binding groove. Our structure–function analyses revealed that the BH3 binding capacity of MCL-1 and its suppression of BAX are impaired by molecular engagement, a phenomenon recapitulated by C286W mutagenic mimicry in vitro and in mouse cells. Thus, we characterize an allosteric mechanism for disrupting the antiapoptotic BH3 binding activity of MCL-1, informing a new strategy for disarming MCL-1 in cancer.   

News & Views by L. Konermann

Pubmed: 27159560

Hydrogen exchange mass spectrometry of related proteins with divergent sequences: A comparative study of HIV-1 Nef allelic variants.
Wales TE, Poe JA, Emert-Sedlak L, Morgan CR, Smithgall TE, Engen JR.

J Am Soc Mass Spectrom. 2016. Jun;27(6):1048-1061.

Hydrogen exchange mass spectrometry can be used to compare the conformation and dynamics of proteins that are similar in tertiary structure.  If relative deuterium levels are measured, differences in sequence, deuterium forward- and back-exchange, peptide retention time and protease digestion patterns all complicate the data analysis.  We illustrate what can be learned from such data sets by analyzing five variants (Consensus G2E, SF2, NL4-3, ELI, and LTNP4) of the HIV-1 Nef protein, both alone and when bound to the human Hck SH3 domain.  Regions with similar sequence could be compared between variants.  While much of the hydrogen exchange features were preserved across the five proteins, the kinetics of Nef binding to Hck SH3 were not the same.  These observations may be related to biological function, particularly for ELI Nef where we also observed an impaired ability to downregulate CD4 surface presentation.  The data illustrate some of the caveats that must be considered for comparison experiments and provide a framework for investigations of other protein relatives, families, and superfamilies with HX MS. 
Pubmed: 27032648

Dynamics of the Tec-family tyrosine kinase SH3 domains.
Roberts JM, Tarafdar S, Joseph RE, Andreotti AH, Smithgall TE, Engen JR, Wales TE.

Protein Sci. 2016. Apr 25;25(4):852-864.

The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins.  X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not.  We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins.  Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx).  The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains.  Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR.  Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling.  The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not.  Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role.  While the exact reason for such dynamics is not clear, such motions can be exploited in intra- and intermolecular binding assays of proteins containing the domains.
Pubmed: 26808198

Tuning a high transmission ion guide to prevent gas-phase proton exchange during H/D exchange MS.
Guttman M, Wales TE, Whittington D, Engen JR, Brown JM, Lee KK.

J Am Soc Mass Spectrom. 2016. Apr;27(4):662-668.

Hydrogen / deuterium exchange (HDX) mass spectrometry (MS) for protein structural analysis has been adopted for many purposes, including biopharmaceutical development. One of the benefits of examining amide proton exchange by mass spectrometry is that it can readily resolve different exchange regimes, as evidenced by either binomial or bimodal isotope patterns. By careful analysis of the isotope pattern during exchange, more insight can be obtained on protein behavior in solution.  However, one must be sure that any observed bimodal isotope patterns are not artifacts of analysis and are reflective of the true behavior in solution. Sample carryover and certain stationary phases are known as potential sources of bimodal artifacts. Here, we describe an additional undocumented source of deuterium loss resulting in artificial bimodal patterns for certain, highly charged peptides. We demonstrate that this phenomenon is predominantly due to gas-phase proton exchange between peptides and bulk solvent within the initial stages of high-transmission conjoined ion guides. Minor adjustments of the ion guide settings, as reported here, eliminate the phenomenon without sacrificing signal intensity. Such gas-phase deuterium loss should be appreciated for all HDX-MS studies using such ion optics, even for routine studies not focused on interpreting bimodal spectra.
Pubmed: 26810432

Dynamic allostery mediated by a conserved tryptophan in the Tec family kinases.
Chopra N, Wales TE, Joseph RE, Boyken SE, Engen JR, Jernigan RL, Andreotti AH.
PLoS Comput Biol. 2016. Mar 24;12(3):e1004826.

Bruton’s tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues.  In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix.  We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.
Pubmed: 27010561

Icon for HighWire
Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome.
Shi Y, Chen X, Elsasser S, Stocks BB, Tian G, Lee B-H, Shi Y, Zhang N, de Poot SAH, Tuebing F, Sun S, Vannoy J, Tarasov SG, Engen JR, Finley D, Walters KJ
Science. 2016. Feb 19;351(6275):aad9421.

Hundreds of pathways for degradation converge at ubiquitin recognition by a proteasome. Here, we found that the five known proteasomal ubiquitin receptors are collectively nonessential for ubiquitin recognition and identified a sixth receptor, Rpn1, in yeast. A site (T1) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like (UBL) domains of substrate shuttling factors. T1 structures with monoubiquitin or lysine 48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for lysine 48–linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site (T2) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus, a two-site recognition domain intrinsic to the proteasome uses homologous ubiquitin and/or UBL-class ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme.
Pubmed: 26912900

Click here to read
Structural stability and local dynamics in disease-causing mutants of human Apolipoprotein A-I: What makes the protein amyloidogenic?
Das M, Wilson CJ, Mei X, Wales TE, Engen JR, Gursky O.
J Mol Biol. 2016. Jan 29;428(2ptB):449-462.

ApoA-I, the major protein of plasma high-density lipoprotein, removes cellular cholesterol and protects against atherosclerosis. ApoA-I mutations can cause familial amyloidosis, a life-threatening disease wherein N-terminal protein fragments form fibrils in vital organs. To unveil the protein misfolding mechanism and to understand why some mutations cause amyloidosis while others do not, we analyzed the structure, stability and lipid-binding properties of naturally occurring mutants of full-length human apoA-I causing either amyloidosis (G26R, W50R, F71Y, L170P) or aberrant lipid metabolism (L159R). Global and local protein conformation and dynamics in solution were assessed by circular dichroism, fluorescence, and hydrogen-deuterium exchange mass spectrometry. All mutants showed increased deuteration in residues 14-22, supporting our hypothesis that decreased protection of this major amyloid “hot spot” can trigger protein misfolding. In addition, L159R showed local helical unfolding near the mutation site, consistent with cleavage of this mutant in plasma to generate the labile 1-159 fragment. Together, the results suggest that reduced protection of the major amyloid “hot spot”, combined with structural integrity of the native helix-bundle conformation, shift the balance from protein clearance to b-aggregation. A delicate balance between the overall structural integrity of a globular protein and the local destabilization of its amyloidogenic segments may be a fundamental determinant of this and other amyloid diseases. Furthermore, mutation-induced conformational changes observed in the helix bundle, which comprises N-terminal 75% of apoA-I, and its flexible C-terminal tail suggest the propagation of structural perturbations to distant sites via an unexpected template-induced ensemble-based mechanism, challenging the classical structure-based view.
Pubmed: 26562506