Lawrence McIntosh


Research Interests

Protein structure-dynamics-function
Regulation of gene expression
Signal transduction
Enzyme mechanisms

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Research Methodology

Biophyical chemistry
NMR spectroscopy


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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Nov 2020)
Insights into the inhibition of ETV6 PNT domain polymerization (2020)

ETV6 is a modular transcriptional repressor for which head-to-tail polymerization of its PNT (or SAM) domain facilitates cooperative binding to tandem DNA sites by its ETS domain. Chromosomal translocations frequently fuse the ETV6 PNT domain to the catalytic domain of one of several receptor protein tyrosine kinases. The resulting chimeric oncoproteins undergo ligand-independent self-association, auto-phosphorylation, and aberrant stimulation of downstream signaling pathways, thereby leading to a diverse range of cancers. Inhibition of PNT domain polymerization through mutations renders the chimeric proteins non-oncogenic. This indicates that a small molecule inhibitor of polymerization could be a viable therapeutic against many ETV6-linked cancers. Protein-protein interactions are challenging to disrupt with small molecules, and thus I followed a multi-pronged approach for lead compound discovery. In Chapter 2, I describe work to obtain structural, dynamic, and thermodynamic insights into the PNT domain and its self-association properties. To this end, I characterized monomeric and heterodimeric forms of the PNT domain using nuclear magnetic resonance spectroscopy, X-ray crystallography, molecular dynamics simulations, amide hydrogen exchange, and alanine scanning mutagenesis in conjunction with surface plasmon resonance binding studies. Collectively, these studies defined “hot spot” regions critical to the PNT domain self-association interface.In Chapter 3, I discuss efforts undertaken to find inhibitors of ETV6 PNT domain polymerization using two high-throughput cellular approaches – a split luciferase reporter assay and a modified yeast two-hybrid assay – and a computational approach with the Bristol University Docking Engine (BUDE). Over 75 lead compounds from these assays were tested for binding to the PNT domain through NMR spectroscopy. None were found to bind to the PNT domain or inhibit its self-association. However, lessons learned from these screening assays may facilitate future high-throughput screening for or rational design of therapeutics that act against ETV6 oncoproteins by disrupting PNT domain polymerization.

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Characterizing labile protons by NMR spectroscopy (2019)

Labile hydrogens in proteins, including those on ionizable functional groups, undergo rapid exchange with water and thus are typically difficult to characterize by current techniques in structural biology. Their properties are often inferred from biophysical arguments rather than direct experimental determination. Here I present new methods for studying these hydrogens by NMR spectroscopy using two glycoside hydrolases as model protein systems. Chapter 2 centres on characterizing the hydroxyl protons of serine and threonine residues in proteins by NMR spectroscopy. Using auxtrophic E. coli strains, I produced Bacillus circulans xylanase (BcX) selectively labeled with ¹³C/¹⁵N-serine or ¹³C/¹⁵N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed using long-range heteronuclear scalar correlation experiments. Their dihedral angle-dependent scalar couplings with adjacent sidechain protons were determined via a quantitative ¹³C/1¹⁵N-filtered spin-echo difference experiment. The hydrogen exchange kinetics of these hydroxyls were measured using a ¹³C/¹⁵N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provided insights into the structural and dynamic properties of this model protein. Chapter 3 characterizes a mutant of T4 phage lysozyme (T4L) with an altered catalytic mechanism due to the substitution of residue Thr26 with a histidine (T26H). It has been proposed that T26H-T4L hydrolyzes peptidoglycan via a double displacement mechanism with His26 serving the unusual role of a nucleophile. To gain further insights into this or alternative mechanisms, I used NMR spectroscopy to measure the acid dissociation constants (pKa values) and/or ionization states of all the Asp, Glu, His, and Arg residues in the T4L mutant. Most notably, the pKa value of the proposed nucleophile His26 is 6.8 ± 0.1, whereas that of the general acid Glu11 is 4.7 ± 0.1. If the proposed mechanism holds true, then T26H-T4L follows a reverse protonation pathway where only a minor population of protein is in its catalytically competent ionization state (His26 deprotonated and Glu11 protonated). I also demonstrated that all arginines in T26H-T4L, including the active site Arg145, are positively charged under neutral pH. This stands in contrast to a recent neutron crystallographic study of T26H-T4L in which, perplexingly, Arg145 was proposed to have a deprotonated guanidine sidechain.

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Biophysical insights into the regulatory and DNA-binding mechanisms of the eukaryotic transcription factors Pax5 and Ets1 (2018)

Transcription factors are proteins that bind at promoter and enhancer sites to regulate gene expression. In this thesis, I used NMR spectroscopy and other methods to investigate the structural and biophysical basis of DNA binding by two eukaryotic transcription factors that are crucial in the development of lymphocytes, Pax5 and Ets1. In chapter 2, I describe how the two subdomains comprising the bipartite DNA-binding Paired domain of Pax5 cooperate to mediate transcriptional regulation. The N-terminal subdomain recognizes DNA sequences in a highly specific manner, whereas the C-terminal subdomain shows little sequence discrimination. The more rigid C-terminal subdomain binds DNA primarily though non-specific electrostatic interactions. In contrast, association with specific DNAs by the dynamic N-terminal subdomain involves relatively large and compensating changes in enthalpy and entropy that point to structural rearrangements upon binding. I propose that the distinct behaviors of the subdomains allow the Pax5 protein to rapidly scan non-specific genomic DNA while retaining specificity for functional regulatory sites. In chapter 3, I expand our understanding of the structural and thermodynamic basis of Ets1 autoinhibition. Previously it was reported that an intrinsically disordered serine-rich region (SRR) interacts transiently with the adjacent ETS domain to attenuate DNA binding. Although forming a dynamic fuzzy complex, I was able to use NMR spectroscopy and X-ray crystallography to provide a detailed mechanism for this inhibitory interaction. In particular, I exploited a trans peptide system to show that the SRR uses a combination of electrostatic and hydrophobic-driven interactions to sterically block the ETS domain DNA-binding interface. I also show how phosphorylation of the SRR strengthens its association with the ETS domain. Altogether, these results explain how the activity of Ets1 is regulated at the level of DNA binding through post-translational modifications that impinge upon the SRR.

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Protein phase separation by the ABC transporter Rv1747 of Mycobacterium tuberculosis (2018)

The Mycobacterium tuberculosis ABC transporter Rv1747 is a putative exporter of cell wall biosynthesis intermediates. Rv1747 has a cytoplasmic regulatory module consisting of two pThr-interacting Forkhead-associated (FHA) domains connected by a conformationally disordered linker with two phospho-acceptor threonines (pThr). In chapter 2, I report the structures of FHA-1 and FHA-2 determined by X-ray crystallography and NMR spectroscopy, respectively. Relative to the canonical 11-strand beta-sandwich FHA domain fold of FHA-1, FHA-2 is circularly permuted and lacking one beta-strand. Nevertheless, the two share a conserved pThr-binding cleft. FHA-2 is less stable and more dynamic than FHA-1, yet binds model pThr peptides with moderately higher affinity (~ 50 uM versus 500 uM equilibrium dissociation constants). Based on NMR relaxation and chemical shift perturbation measurements, when joined within a polypeptide chain, either FHA domain can bind either linker pThr to form intra- and intermolecular complexes. Protein phase separation has been recently shown to be a fundamental mechanism underlying the clustering of some proteins at the eukaryotic cell membrane. In chapter 3, I show that upon multi-site phosphorylation of the linker by several Mtb serine/threonine protein kinases including PknF, the isolated regulatory module readily multimerizes and phase separates into dynamic liquid droplets with diagnostic properties similar to those exhibited by eukaryotic proteins. The process is reversed by the sole Mtb serine/threonine phosphatase PstP. In the absence of phosphorylation, the Rv1747 regulatory module can still undergo phase separation, albeit at higher protein concentrations and into droplets with more fluid properties. This points to a synergy between specific FHA-pThr binding and additional weak association of the ID linker and/or the FHA domains leading to the pre-requisite multivalent interactions for demixing. Droplet formation of the regulatory module was replicated in a pseudo-two-dimensional system on model supported lipid bilayers. Potential clusters of Rv1747 were also detected in Mtb using ultra-high resolution Direct Stochastic Reconstruction Microscopy (dSTORM). This is the first reported example of phase separation by both a bacterial protein and an ABC transporter, and suggests possible mechanisms for the regulation of Rv1747 within Mtb. I hypothesize that the tunable, phosphorylation-dependent multimerization described here regulates Rv1747 transporter activity.

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Understanding ETS transcription factors: from ordered domains to disordered sequences (2017)

ETS (E26 transformation specific) transcription factors play critical roles in regulating cellular growth, development and differentiation. They share a conserved ETS domain that interacts with specific DNA sequences and a subset of ETS proteins also contain a PNT domain responsible for protein partnerships. My research initially focused on the PNT domain of Drosophila Pointed-P2, with the goal of understanding the impact of phosphorylation on the activation of gene expression. Using a battery of NMR spectroscopic approaches, I demonstrated that the Pointed-P2 PNT domain contains a dynamic N-terminal helix H0 appended to a core conserved five-helix bundle. This helix must be displaced to allow docking of the PNT domain with the ERK2 MAP kinase Rolled, which in turn phosphorylates three N-terminal phosphoacceptor sites. The second part of my thesis focuses on three members of the ETS family called the ETV1/4/5 sub-group: ETV1 (Er81), ETV5 (Erm), and ETV4 (PEA3 (polyoma enhancer activator 3)). Using an extensive set of ETV4 deletion fragments, the DNA binding autoinhibitory sequences at both N- and C-terminal to the ETS domain were identified. Through detailed NMR spectroscopic studies, I confirmed that the inhibitory sequences are predominantly disordered and transiently interact with a coarsely defined surface on the ETS domain. This surface overlaps the DNA-recognition interface, thus indicating a steric mechanism of autoinhibition. Overall, my studies help define the molecular mechanisms underlying ETV1/4/5 factors autoinhibition, and may inspire new anti-cancer strategies.Finally, I also investigated the stability and dynamics of several uninhibited ETS domains, including ETV4, PU.1, Ets1, and ETV6. Using NMR spectroscopy, I determined the structure of the PU.1 ETS domain and identified an appended, C-terminal helix. Similar to Ets1 and ETV6, the DNA-recognition helix H3 of ETV4 and PU.1 are dynamic as evidenced by amide hydrogen exchange (HX). I also utilized molecular dynamics simulations to map the motions of the four ETS domains and identified several critical pathways that may impact their stabilities and possibly, the DNA-binding abilities. Overall, the data presented in my thesis will provide further understanding of the structure and regulation of the ETS transcription factors.

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Structural characterization of DNA binding and autoinhibition by the Ets1 transcription factor (2015)

Ets1 belongs to the ETS transcription factor family and plays key roles in regulating eukaryotic gene expression. The affinity of the Ets1 for its cognate DNA sites is autoinhibited by an intrinsically disordered serine-rich region (SRR) and an appended helical inhibitory module (IM). Through transient interactions, the SRR both sterically blocks the ETS domain and allosterically stabilizes the IM to modulate DNA-binding affinity. Calmodulin-dependent kinase II phosphorylation of five serines within the SRR progressively reinforces autoinhibition in response to calcium signaling.Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine/tyrosine (ϕ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-ϕ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforcesautoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively-charged DNA recognition interface and an adjacentregion of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence that integrates cellular signaling and gene expression.We also investigated by NMR spectroscopy the interaction of Ets1 with specific and nonspecific oligonucleotides. Upon binding DNA, helices HI-1 and HI-2 of the IM unfold. Thus, autoinibition does not impart DNA-binding specificity. Using amide chemical shift perturbation mapping, we also show that Ets1 binds both specific and non-specific oligonucleotides through its canonical ETS domain interface. However, the non-specific complex is formed by weak anddynamic electrostatic interactions, whereas the specific complex involves well-ordered hydrogen bonds and salt bridges. In support of this conclusion, five lysine sidechains are protected from rapid hydrogen exchange upon binding of specific DNA, whereas only one is stabilized in the non-specific complex. Overall, these data are consistent with Ets1 rapidly finding specific DNA sites within the genome via facilitated diffusion (sliding and hopping) within a vast background of non-specific sequences.

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Investigating the Enzymatic Mechanisms of the Inverting and Retaining Glycosyltransferases by NMR Spectroscopy (2012)

The overall goal of this thesis was to investigate the structures and enzymatic mechanisms of glycosyltransferases using NMR spectroscopy and enzyme kinetic measurements. The bifunctional sialyltransferase CstII from Campylobacter jejuni and the α-1,4-galactosyltransferase LgtC from Neisseria meningitidis were chosen to be the model inverting and retaining enzymes, respectively. By systematically introducing point mutations at the subunit interfaces of CstII, two active monomeric variants were obtained and characterized. In contrast to the wild-type tetramer, the monomeric CstII variants yielded good quality amide ¹H/¹⁵N-HSQC and methyl-TROSY NMR spectra. However, the absence of signals from approximately one half of the amides in the ¹H/¹⁵N-HSQC spectra of both monomeric forms suggests that the enzyme undergoes substantial conformational exchange on a msec-µsec time-scale. The histidine pKa values of CstII-F121D in its apo form were measured by monitoring the pH-dependent chemical shifts of biosynthetically incorporated [¹³Cε¹]-histidine. Consistent with its proposed catalytic role, the site-specific pKa value ~ 6.6 for His188 matches the apparent pKa value ~ 6.5 governing the pH-dependence of kcat/Km for CstII towards CMP-Neu5Ac. The enzymatic mechanism of the retaining glycosyltransferase LgtC appears to involve a “front-side attack” SNi or SNi-like mechanism with a short-lived oxocarbenium-phosphate ion pair intermediate. Furthermore, based upon X-ray crystallographic studies, two flexible loops were proposed to become ordered over the active site of LgtC upon sugar donor binding. Accordingly, NMR spectroscopy was used to investigate the dynamic properties of the enzyme with an emphasis on delineating the possible roles of these motions. The amide ¹H/¹⁵N-TROSY-HSQC and methyl-TROSY spectra of LgtC were partially assigned using a variety of NMR spectroscopic approaches, combined with mutagenesis of all the isoleucine residues. More than the expected number of methyl signals was observed, indicating that LgtC adopts multiple conformational states in equilibrium on a seconds time-scale, and that their relative populations change upon mutation and substrate binding. Furthermore, relaxation dispersion studies indicated substantial msec-µsec time-scale motions of methyl groups both within and distal to the active site in apo and substrate-bound forms of LgtC. Thus LgtC exhibits a range of dynamic behaviours potentially linked to its catalytic function. They were studies in this thesis using NMR spectroscopy and kinetic studies.

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Structural and Functional Characterization of the DAXX N-Terminal Helix Bundle and SUMO Interaction Motifs (2010)

The Fas death-domain associated (DAXX) protein was first discovered as an intermediary of a FADD-independent apoptosis signaling pathway. However, subsequent studies have established it as an important player in both transcription and cell cycle regulation.In this thesis, the first structural characterization of DAXX is presented. Sequence alignment and secondary structure prediction algorithms were used to define a number of constructs of DAXX. The C-terminal ~l/3 of DAXX was found to be intrinsically disordered, whereas a well-defined folded domain was identified near its N-terminus. NMR spectroscopy was used to solve the three-dimensional structure of this domain, and to characterize its dynamic behavior. The calculated structural ensemble consists of five helices, and hence is named the DAXX HelixBundle (DHB) domain. This domain has a very different topology to the Sin3 PAH domains, which until now have been used as a model for this region of DAXX.Rassfl C, an important tumor suppressor, was reported recently to interact with DAXX via the DHB domain. This interaction was linked to mitosis progression, and has potential implications in the treatment of cancer. The NMR-derived ensemble of the complex of the DHB domain with an N-terminal fragment of Rassf1 C revealed a short amphipathic a-helix filling the cleft between helices 2 and 5 of the DHB domain. Both hydrophobic and electrostatic interactions mediated complex formation. This structural characterization explains the observed in vivo interaction and provides clues as to how the binding might be regulated.Additionally, two SUMO-interacting motifs at the termini of DAXX, SIM-N and SIM-C, were characterized. Their interactions with SUMO- 1 and SUMO-2 were examined structurally and thermodynamically using NMR spectroscopy. SIM-N was also found to bindintramolecularly to the DHB domain and SIM-C to mediate the interaction of DAXX with thesumoylated Ets- 1 transcription factor. Importantly, the latter did not involve any direct contactsbetween DAXX and Ets- 1, but rather derived from the non-covalent binding of DAXX SIM-C to SUMO- 1, which in turn was covalently linked in a “beads-on-a-string” fashion to Ets- 1. These results provide new insights into the binding mechanisms and biological roles of DAXX-SUMO interactions.

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Structural foundation for transcriptional regulation by Ets1 and CBP (2010)

Using cell-based assays and biophysical measurements, we have defined the mechanism by which Ras/MAP kinase signaling enhances Ets1 regulated gene expression via phosphorylation-enhanced recruitment of the transcriptional co-activator CBP. As confirmed by ³¹P/¹³C-NMR experiments, the MAP kinase ERK2 phosphorylates Thr38 and Ser41 within the unstructured region of Ets1, immediately N- terminal to the PNT domain. The NMR-derived structure of residues 29-138 of Ets1 revealed that the PNT domain is composed of a core four-helix bundle (H2-H5), also known as the SAM fold, appended with two additional helices (H0-H1). Most importantly, helix H0 is only marginally stable as shown by various NMR methods, including chemical shift, amide hydrogen exchange, and ¹⁵N relaxation analyses.Dual phosphorylation of Ets1 perturbs a "closed-open" conformational equilibrium of the PNT domain, displacing the dynamic helix H0 from the core bundle. These modifications increase the affinity of Ets1 for the TAZ1 (or CH1) domain of CBP by ~30 fold as measured with isothermal titration calorimetry (Kd ~ 60 to 2 μM). NMR-monitored titration experiments mapped the interaction surfaces of the TAZ1 domain and Ets1, the latter encompassing both the phosphoacceptors and PNT domain, also showing sensitivity to ionic strength. Charge complementarity of these surfaces indicates that electrostatic forces act in concert with the conformational equilibrium to mediate phosphorylation effects.We conclude that the dynamic helical elements of Ets1, appended to a conserved structural core, constitute a "phospho-switch" to direct Ras/MAPK signaling to downstream changes in gene expression. This detailed structural and mechanistic information illustrates an evolutionary development within a gene family to increase the capacity for biological regulation.We also discovered that the CBP TAZ1 domain associates intramolecularly with residues 28-82 in its N-terminal nuclear receptor interacting domain (NRID). NMR studies indicated that the NRID undergoes a coil-helix conformational transition upon binding the same interface on TAZ1 as recognized by many transcription factor partners. This led us to hypothesize that CBP is regulated by an auto-inhibitory mechanism. In support of this model, affinity of the hypoxia inducible factor HIF-1α for TAZ1 is reduced competitively by the presence of the NRID.

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Master's Student Supervision (2010 - 2018)
Dissecting the mechanism of ETV6 polymerization (2014)

ETV6 (or TEL), a member of the ETS family of eukaryotic transcription factors, normally functions as a transcriptional repressor and putative tumor suppressor. ETV6 is modular, containing a SAM (or PNT) domain and a DNA-binding ETS domain joined by a flexible linker sequence. The ETV6 SAM domain self-associates in a head-to-tail fashion, forming helical polymers proposed to generate extended repressive complexes at target DNA sites. ETV6 is also frequently involved in chromosomal translocations yielding unregulated chimeric oncoproteins with the SAM domain fused to the catalytic domain of a tyrosine receptor kinase such as NTRK3. Cellular transformation likely results from SAM domain-mediated polymerization and constitutive activation of the kinase domain. In the case of the ETV6- NTRK3 fusion (EN), this transformation is linked to congenital fibrosarcomas. Our goal is to investigate via mutations within its SAM domain, the thermodynamic and dynamic mechanisms underlying the altered transformation properties of ETV6-NTRK3. These studies have been carried out using monomeric variants of the isolated SAM domains with "head" or "tail" point mutations that prevent self-association, yet allow for formation of a mixed dimer with a native binding interface. Specifically, we used a combination of NMR spectroscopy and isothermal titration calorimetry to study the effects of additional mutations on their dimerization. Consistent with its involvement in a crystallographically-observed interdomain salt bridge, mutation of Lys99 was found to weaken the association of ETV-SAM monomers in solution, and to disrupt cellular transformation by EN. This supports the role of the SAM domain self-association in the activation of ETV6-NTRK3, and helps define the mechanisms underlying cellular transformation by similar chimeric oncoproteins.

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