Predicting and testing determinants of histidine-kinase functions by leveraging protein sequence information

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, February 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis. "September 2012."Includes bibliographical references.All cells sense and respond to their environments using signal transduction pathways. These pathways control a sweeping variety of cellular processes across the domains of life, but the pathways are often built from a small, shared set of protein domains. At the core of tens of thousands of signal transduction networks in bacteria is a pair of proteins, a histidine kinase and a response regulator. Upon receiving an input signal, a histidine kinase autophosphorylates and then catalyzes transfer of its phosphoryl group to a cognate response regulator, which often activates a transcriptional response. Bacteria typically encode dozens of kinases and regulators, and the kinases function as dimers in all known examples. This dimeric state raises two functional questions. Do histidine kinases specifically form dimers? Once a kinase has dimerized, does a chain in the dimer phosphorylate itself (cis) or its partner chain (trans)? Specific kinase dimerization is likely important to avoid detrimental crosstalk between separate signaling pathways, and how autophosphorylation occurs is central to kinase activity. In my thesis, I have taken biochemical and evolutionary approaches to identify molecular determinants for both dimerization specificity and autophosphorylation. To study dimerization specificity, I developed an in vitro binding assay to measure kinase dimerization, and I then showed that a paralogous pair of kinases from E. coli specifically formed homodimers over heterodimers. Residues important for dimerization specificity were predicted by measuring amino acid coevolution within kinases, which leverages the enormous amount of sequence information available for the kinase family. Experimental verification of these predictions showed that a set of residues at the base of the kinase dimerization domain was sufficient to establish homospecificity. This same region of the kinase, in particular the loops at the base of the kinase dimer, was also important for determining autophosphorylation mechanism. Recent work showed that kinases could autophosphorylate either in cis or in trans, and I found that a trans kinase could be made to autophosphorylate in cis by replacing its loop with the loop from a cis kinase. I also found that two sets of orthologs, despite having significantly diverged loop sequences, had conserved their autophosphorylation mechanisms. This raised the possibility that kinase loops may be under selection to maintain the same autophosphorylation mechanism.by Orr Ashenberg.Ph.D

Robust protein-protein interactions in crowded cellular environments

The capacity of proteins to interact specifically with one another underlies our conceptual understanding of how living systems function. Systems-level study of specificity in protein-protein interactions is complicated by the fact that the cellular environment is crowded and heterogeneous; interaction pairs may exist at low relative concentrations and thus be presented with many more opportunities for promiscuous interactions compared to specific interaction possibilities. Here we address these questions using a simple computational model that includes specifically designed interacting model proteins immersed in a mixture containing hundreds of different unrelated ones; all of them undergo simulated diffusion and interaction. We find that specific complexes are quite robust to interference from promiscuous interaction partners, only in the range of temperatures Tdesign>T>Trand. At T>Tdesign specific complexes become unstable, while at T<Trand formation of specific complexes is suppressed by promiscuous interactions. Specific interactions can form only if Tdesign>Trand. This condition requires an energy gap between binding energy in a specific complex and set of binding energies between randomly associating proteins, providing a general physical constraint on evolutionary selection or design of specific interacting protein interfaces. This work has implications for our understanding of how the protein repertoire functions and evolves within the context of cellular systems

Determinants of Homodimerization Specificity in Histidine Kinases

Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and RstB, its closest paralog in Escherichia coli. Direct binding studies showed that the cytoplasmic domains of these proteins each form specific homodimers in vitro. Using a series of chimeric proteins, we identified specificity determinants at the base of the four-helix bundle in the dimerization and histidine phosphotransfer domain. Guided by molecular coevolution predictions and EnvZ structural information, we identified sets of residues in this region that are sufficient to establish homospecificity. Mutating these residues in EnvZ to the corresponding residues in RstB produced a functional kinase that preferentially homodimerized over interacting with EnvZ. EnvZ and RstB likely diverged following gene duplication to yield two homodimers that cannot heterodimerize, and the mutants we identified represent possible evolutionary intermediates in this process.National Institutes of Health (U.S.) (Award GM067681)National Science Foundation (U.S.) (CAREER Grant)National Science Foundation (U.S.). Graduate Research Fellowshi

Spatial tethering of kinases to their substrates relaxes evolutionary constraints on specificity

Signal transduction proteins are often multi-domain proteins that arose through the fusion of previously independent proteins. How such a change in the spatial arrangement of proteins impacts their evolution and the selective pressures acting on individual residues is largely unknown. We explored this problem in the context of bacterial two-component signalling pathways, which typically involve a sensor histidine kinase that specifically phosphorylates a single cognate response regulator. Although usually found as separate proteins, these proteins are sometimes fused into a so-called hybrid histidine kinase. Here, we demonstrate that the isolated kinase domains of hybrid kinases exhibit a dramatic reduction in phosphotransfer specificity in vitro relative to canonical histidine kinases. However, hybrid kinases phosphotransfer almost exclusively to their covalently attached response regulator domain, whose effective concentration exceeds that of all soluble response regulators. These findings indicate that the fused response regulator in a hybrid kinase normally prevents detrimental cross-talk between pathways. More generally, our results shed light on how the spatial properties of signalling pathways can significantly affect their evolution, with additional implications for the design of synthetic signalling systems.National Science Foundation (U.S.) (CAREER Award)National Science Foundation (U.S.). Graduate Research Fellowship Progra

Systematic Dissection and Trajectory-Scanning Mutagenesis of the Molecular Interface That Ensures Specificity of Two-Component Signaling Pathways

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk

Single-cell meta-analysis of SARS-CoV-2 entry genes across tissues and demographics

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention

Decoding human fetal liver haematopoiesis.

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.We acknowledge funding from the Wellcome Human Cell Atlas Strategic Science Support (WT211276/Z/18/Z); M.H. is funded by Wellcome (WT107931/Z/15/Z), The Lister Institute for Preventive Medicine and NIHR and Newcastle-Biomedical Research Centre; S.A.T. is funded by Wellcome (WT206194), ERC Consolidator and EU MRG-Grammar awards and; S.B. is funded by Wellcome (WT110104/Z/15/Z) and St. Baldrick’s Foundation; E.L. is funded by a Wellcome Sir Henry Dale and Royal Society Fellowships, European Haematology Association, Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute and BBSRC

Helix Bundle Loops Determine Whether Histidine Kinases Autophosphorylate in cis or in trans

Bacteria frequently use two-component signal transduction pathways to sense and respond to environmental and intracellular stimuli. Upon receipt of a stimulus, a homodimeric sensor histidine kinase autophosphorylates and then transfers its phosphoryl group to a cognate response regulator. The autophosphorylation of histidine kinases has been reported to occur both in cis and in trans, but the molecular determinants dictating which mechanism is employed are unknown. Based on structural considerations, one model posits that the handedness of a loop at the base of the helical dimerization domain plays a critical role. Here, we tested this model by replacing the loop from Escherichia coli EnvZ, which autophosphorylates in trans, with the loop from three PhoR orthologs that autophosphorylate in cis. These chimeric kinases autophosphorylated in cis, indicating that this small loop is sufficient to determine autophosphorylation mechanism. Further, we report that the mechanism of autophosphorylation is conserved in orthologous sets of histidine kinases despite highly dissimilar loop sequences. These findings suggest that histidine kinases are under selective pressure to maintain their mode of autophosphorylation, but they can do so with a wide range of sequences.National Institutes of Health (U.S.) (Award GM067681)National Science Foundation (U.S.) (CAREER Grant)National Science Foundation (U.S.). Graduate Research Fellowshi

Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA

<div><p>The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors.</p></div

A mutation at site 51 in NP increases MxA sensitivity as measured by viral competition.

<p>We used reverse genetics to generate viruses carrying the wildtype NP, the D51N mutation, or a synonymous mutation at site 51. Each virus was generated in duplicate. We mixed the wildtype virus with one of the mutant viruses, infected MxA-expressing and non-expressing cells, and measured mutant frequencies after 10 and 54 hours. The plots show the frequency of the wildtype D51 variant relative to the N51 amino-acid mutant or the Dsyn51 synonymous mutant at each timepoint. In both replicates of the competition, the wildtype D51 variant was strongly favored over the N51 mutant in MxA-expressing cells. We targeted a 1:1 ratio of infectious particles in our initial inoculum, but this ratio was impossible to verify by direct sequencing since sequencing cannot distinguish infectious from non-infectious virions.</p