Structural compliance: A new metric for protein flexibility

Proteins are the active players in performing essential molecular activities throughout biology, and their dynamics has been broadly demonstrated to relate to their mechanisms. The intrinsic fluctuations have often been used to represent their dynamics and then compared to the experimental B-factors. However, proteins do not move in a vacuum and their motions are modulated by solvent that can impose forces on the structure. In this paper, we introduce a new structural concept, which has been called the structural compliance, for the evaluation of the global and local deformability of the protein structure in response to intramolecular and solvent forces. Based on the application of pairwise pulling forces to a protein elastic network, this structural quantity has been computed and sometimes is even found to yield an improved correlation with the experimental B-factors, meaning that it may serve as a better metric for protein flexibility. The inverse of structural compliance, namely the structural stiffness, has also been defined, which shows a clear anticorrelation with the experimental data. Although the present applications are made to proteins, this approach can also be applied to other biomolecular structures such as RNA. This present study considers only elastic network models, but the approach could be applied further to conventional atomic molecular dynamics. Compliance is found to have a slightly better agreement with the experimental B-factors, perhaps reflecting its bias toward the effects of local perturbations, in contrast to mean square fluctuations. The code for calculating protein compliance and stiffness is freely accessible at https://jerniganlab.github.io/Software/PACKMAN/Tutorials/compliance

Characterizing and Predicting Protein Hinges for Mechanistic Insight

The functioning of proteins requires highly specific dynamics, which depend critically on the details of how amino acids are packed. Hinge motions are the most common type of large motion, typified by the opening and closing of enzymes around their substrates. The packing and geometries of residues are characterized here by graph theory. This characterization is sufficient to enable reliable hinge predictions from a single static structure, and notably, this can be from either the open or the closed form of a structure. This new method to identify hinges within protein structures is called PACKMAN. The predicted hinges are validated by using permutation tests on B-factors. Hinge prediction results are compared against lists of manually-curated hinge residues, and the results suggest that PACKMAN is robust enough to reproduce the known conformational changes and is able to predict hinge regions equally well from either the open or the closed forms of a protein. A group of 167 protein pairs with open and closed structures has been investigated Examples are shown for several additional proteins, including Zika virus non-structured (NS) proteins where there are 6 hinge regions in the NS5 protein, 5 hinge regions in the NS2B bound in the NS3 protease complex and 5 hinges in the NS3 helicase protein. Results obtained from this method can be important for generating conformational ensembles of protein targets for drug design. PACKMAN is freely accessible at (https://PACKMAN.bb.iastate.edu/)

In vitro and in vivo studies of Bruton tyrosine kinase (BTK) mutations & inhibition

Bruton tyrosine kinase (BTK) is a non-receptor protein kinase that belongs to the TEC family kinases. It plays an important role in the B-cell receptor signaling pathway (BCR) and its pharmacological inhibition has been demonstrated as an effective strategy for the treatment of B-cell malignancies. Ibrutinib, acalabrutinib and zanubrutinib are small molecules and irreversible BTK binders that have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of several B-cell malignancies. Irreversible inhibitors block BTK catalytic activity by covalently binding to the cysteine (C) 481 located in the kinase domain. Mutations at this residue abrogate the possibility of forming a covalent bond, thereby decreasing the efficacy of the inhibitor. The most common mutation found in treated patients is the cysteine-481 to serine substitution (C481S). However, other less frequent substitutions have also been identified, such as, T474I and T474S substitutions in the BTK gatekeeper residue or PLCg2 gain-of-function substitutions e.g. S707Y and R665W. In paper I we studied a novel C481S knock-in mouse model. Our analysis of these mice reveled no phenotype alterations, as compared to wild-type mice, and demonstrated that C481S substitution has no detectable effect on BTK´s function or on the development of hematopoietic cells. We demonstrated that isolated B-lymphocytes carrying C481S were resistant to irreversible but sensitive to reversible BTK inhibitors (BTKis). This was achieved by analyzing BTK catalytic activity, cell-viability and expression of cell activation markers. Additionally, we confirmed that irreversible BTKis impaired T-lymphocyte activation in a BTK independent manner. This demonstrates the potential of this mouse model to be used in the study of BTKindependent, both therapeutic and adverse, effects caused by irreversible BTKis. Resistance to BTKis has become one of the most critical concerns in long term ibrutinib treated patients. The cause of the resistance to irreversible BTKis is less frequently associated to the gatekeeper residue, in contrast what is observed for other kinase inhibitors such as the fusionprotein BCR-ABL inhibitor imatininb or the EGFR inhibitor gefitinib. In paper II we aimed to understand the role of gatekeeper and combined gatekeeper/C481 BTK variants in the resistance to reversible and irreversible BTKis. We evaluated protein expression, catalytic activity and susceptibility to BTKis of 16 BTK single and double variants. We found that double T474I/C481S, T474M/C481S and T474M/C481T variants were insensitive to ³16 fold irreversible inhibitor pharmacological serum concentration. On the other hand, reversible BTKis showed a variable inhibition pattern. RN486 seemed to have highest therapeutic potential for patients that develop resistance to combined gatekeeper/C481 BTK variants

PROGRESS TOWARDS THE STRUCTURAL BASIS OF TEC-FAMILY KINASE ACTIVATION BY HIV-1 NEF

HIV-1 Nef is a viral accessory factor that is essential for virus infectivity, host immune evasion and AIDS progression. Nef lacks intrinsic catalytic activity and functions instead via interactions with multiple classes of host cell proteins involved in signal transduction and endocytic trafficking. Nef interacts with the Src-family tyrosine kinases Hck and Lyn through their SH3 domains, resulting in constitutive kinase activity. Nef also binds to select members of the Tec-family of tyrosine kinases, including Btk, Bmx, and Itk, all of which are expressed in HIV-1 target cells. Of particular interest is Itk, which is expressed in CD4+ T cells and is activated by Nef. Selective Itk inhibitors block Nef-dependent enhancement of HIV-1 infectivity and replication, suggesting an important role in the viral life cycle. While the interaction between Itk and Nef has been demonstrated at the plasma membrane in cell-based fluorescence complementation assays, the structural basis of this interaction has not been reported. Like Src-family kinases, Itk has a core region consisting of sequential SH3, SH2 and kinase domains. In addition, Itk has an N-terminal pleckstrin homology (PH) domain important for membrane targeting as well as a Tec homology(TH) region involved in kinase regulation. To explore the structure of the Nef:Tec-family kinase (TFK) complexes, I have created a panel of bacterial expression constructs for the Itk and Btk regulatory region. These include the entire PH-TH-SH3-SH2 region, the SH3-SH2 region, and the isolated SH3 domain, all of which have yielded mg amounts of soluble protein. I have also produced recombinant, N-terminally myristoylated (Myr) Nef in bacteria, a post-translational modification essential for Nef membrane localization in cells. Preliminary Surface Plasmon Resonance (SPR) studies show that Myr-Nef binds membrane bilayers with low µM affinity in a Myr-dependent manner. These proteins will provide the foundation for future structural determination of Nef-TFK complexes by X-ray crystallography as well as the nature of this interaction in lipid bilayers, the physiological site of interaction in HIV-infected cells

Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases

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.This article is published as Chopra, Nikita, Thomas E. Wales, Raji E. Joseph, Scott E. Boyken, John R. Engen, Robert L. Jernigan, and Amy H. Andreotti. "Dynamic allostery mediated by a conserved tryptophan in the Tec family kinases." PLoS computational biology 12, no. 3 (2016): e1004826. doi:10.1371/journal.pcbi.1004826. Posted with permission.</p

Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

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

Molecular determinants regulating Bruton’s tyrosine kinase activity and their mechanism: a combined computational and experimental approach

Understanding allostery in proteins is critical in understanding their unique regulatory mechanisms and this knowledge can be exploited to develop highly specific, targeted therapies. In this dissertation, we have investigated the unique sequence elements that regulate the activity of a protein tyrosine kinase called Bruton’s tyrosine kinase or Btk. Btk is a member of the immune signaling pathway in B-cells and is required for B-cells maturation and function. Lack of a three-dimensional structure of full-length Btk kinase has proved a roadblock in understanding how the domains in Btk interact to shift its conformational equilibrium between active and inactive states. Moreover, in-spite of high homology between the catalytic centers of Btk and other well-studied protein tyrosine kinases such as Src, the regulatory mechanisms of these kinases appear to differ significantly creating an impediment to gaining a complete understanding of the mode of Btk regulation. In pursuit of the aim to identify key sequence and structure motifs that regulate Btk activity, we made use of a range of computational tools to better understand the Btk kinase domain and, when possible, the resulting hypotheses were tested using experimental methods. First, we have identified a specific isoleucine residue, conserved in Btk and related kinases, which functions to stabilize the inactive Btk conformation. We showed that substitution of the conserved isoleucine to leucine shifts the conformational equilibrium of the Btk kinase domain to the active state. Next, we showed how a highly conserved tryptophan, located in a linker region adjacent to the Btk kinase domain, stabilizes the active Btk kinase domain conformation through correlated dynamic motions within the kinase domain itself. Finally, sequence-structure information, combined with information theory and molecular dynamics, was used to identify a specific site in the Btk kinase domain that can be targeted to rescue the kinase activity of Btk in the presence of an inactivating disease causing mutation. The work presented here provides new insights into the regulatory mechanisms in Btk as well as potential allosteric sites in the protein, for which modulators of Btk activity could be developed. There is a growing need for the discovery of such allosteric modulators as Btk has been implicated in immunodeficiency disorders such as X-linked agammaglobulinemia as well as B-cells malignancies and breast and colon cancers. Ultimately, increased knowledge about the molecular mechanisms controlling Btk function should lead to the development of novel Btk activity modulators

Using evolutionary covariance to infer protein sequence-structure relationships

During the last half century, a deep knowledge of the actions of proteins has emerged from a broad range of experimental and computational methods. This means that there are now many opportunities for understanding how the varieties of proteins affect larger scale behaviors of organisms, in terms of phenotypes and diseases. It is broadly acknowledged that sequence, structure and dynamics are the three essential components for understanding proteins. Learning about the relationships among protein sequence, structure and dynamics becomes one of the most important steps for understanding the mechanisms of proteins. Together with the rapid growth in the efficiency of computers, there has been a commensurate growth in the sizes of the public databases for proteins. The field of computational biology has undergone a paradigm shift from investigating single proteins to looking collectively at sets of related proteins and broadly across all proteins. we develop a novel approach that combines the structure knowledge from the PDB, the CATH database with sequence information from the Pfam database by using co-evolution in sequences to achieve the following goals: (a) Collection of co-evolution information on the large scale by using protein domain family data; (b) Development of novel amino acid substitution matrices based on the structural information incorporated; (c) Higher order co-evolution correlation detection. The results presented here show that important gains can come from improvements to the sequence matching. What has been done here is simple and the pair correlations in sequence have been decomposed into singlet terms, which amounts to discarding much of the correlation information itself. The gains shown here are encouraging, and we would like to develop a sequence matching method that retains the pair (or higher order) correlation information, and even higher order correlations directly, and this should be possible by developing the sequence matching separately for different domain structures. The many body correlations in particular have the potential to transform the common perceptions in biology from pairs that are not actually so very informative to higher-order interactions. Fully understanding cellular processes will require a large body of higher-order correlation information such as has been initiated here for single proteins