Leslie Knipling

The Local Electrostatic Environment Determines Cysteine Reactivity of Tubulin

Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [14C]iodoacetamide,N-[14C]ethylmaleimide, or IAEDANS ([5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios ≪1 cysteine/dimer always leads to labeling of 6–7 cysteine residues. These have been identified as Cys-305α, Cys-315α, Cys-316α, Cys-347α, Cys-376α, Cys-241β, and Cys-356β by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347α as the most reactive cysteine of tubulin. Using the 3.5-Å electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 Å of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity.

Direct photoaffinity labeling of tubulin with colchicine.

Ultraviolet irradiation of the [3H]colchicine-tubulin complex leads to direct photolabeling of tubulin with low but practicable efficiency. The bulk (70% to greater than 90%) of the labeling occurs on beta-tubulin and appears early after irradiation, whereas alpha-tubulin is labeled later. The labeling ratio of beta-tubulin to alpha-tubulin (beta/alpha ratio) is reduced by prolonged incubation, prolonged irradiation, urea, high ionic strength, the use of aged tubulin, dilution of tubulin, or large concentrations of colchicine or podophyllotoxin. Glycerol increases the beta/alpha ratio. Limited data with [3H]podophyllotoxin show that it covalently bound with a similar beta/alpha distribution. Vinblastine, on the other hand, exhibits preferential attachment to alpha-tubulin. The possibilities that colchicine binds at the interface between alpha-tubulin and beta-tubulin, that the drug spans this interface, and that both subunits may contribute to the binding site are suggested.

Isolation of microtubule protein from mammalian brain frozen for extended periods of time

Microtubule protein (MTP) may be isolated in good yield from frozen brains by cycles of temperature-dependent polymerization and depolymerization. If the brains are frozen quickly and stored at -70 degrees C, the yield of MTP is stable for a period of at least 2 months and the yield is only slightly decreased after nearly a year. Cow as well as rat brains may be stored in this manner, provided appropriate precautions are taken to ensure rapid freezing of the cow brain. This procedure allows brains to be accumulated over a period of time for MTP isolation at a convenient later date.

Preparation and properties of pure tubulin S

Limited proteolysis of the tubulin dimer (alphabeta) by subtilisin occurs more rapidly with beta than with alpha tubulin. This leads to the formation of an intermediate hybrid dimer, alphabeta(s), before both C termini are cleaved to form tubulin S(alpha(s)beta(s)). The three forms of tubulin usually coexist in subtilisin-treated preparations and such cross-contamination can be reliably detected only by running SDS-polyacrylamide gels well beyond expulsion of the dye front. Previously published preparations have not ruled out such contamination or have formed poorly reversible polymers. Because ion exchange separation incurred substantial protein losses, we have developed a new protocol for rapid preparation of tubulin S (alpha(s)beta(s), free of alphabeta or alphabeta(s)) that is based on proteolysis at low ionic strength. This increases the relative rate of C terminal cleavage of beta tubulin. The product forms sheets, bundles, or rings that are depolymerized by cold, salt, and podophyllotoxin, partially depolymerized by Ca2+, and has a decreased critical concentration for polymerization that can be further decreased by taxol. We have also found a method for forming nearly pure alphabeta(s) dimers by using methods that retard proteolysis of the C terminus of alpha tubulin.

Charge variants of tubulin, tubulin S, membrane-bound and palmitoylated tubulin from brain and pheochromocytoma cells

Isoelectric focusing (IEF) of only approximately 1 microg of rat brain tubulin yields 27-30 distinct charge variants in the pH range of 4.5-5.4 with band separations of 0.01-0.02 pH units as detected by silver staining. Variants can be efficiently transferred from the immobilized gradient strip to polyvinylidene difluoride (PVDF) membranes for reaction with monoclonal antibodies. C-terminal-directed antibodies to alpha- and beta-tubulin yield patterns similar to N-terminal-directed antibodies. Removal of the acidic C-termini with subtilisin to form tubulin S increases the pI values by approximately 1 pH unit, leads to a loss in the isoelectric distinction between the alpha- and beta-tubulin variants seen by N-terminal-directed antibodies, and abolishes reactions with all beta-variants and all but three alpha variants by C-terminal-directed antibodies (TU-04 and TU-14). Many, but not all, of the variants are substrates for autopalmitoylation of rat brain tubulin. The distribution of isoelectric variants differs between cytoplasm and membrane fractions from PC12 pheochromocytoma cells. A potential role for different variants is suggested.

Architecture of the Bacteriophage T4 Activator MotA/Promoter DNA Interaction during Sigma Appropriation

Background: No structure exists of the bacteriophage T4 activator MotA with DNA. Results: Using FeBABE, physical models, and ICM Molsoft, we determined how MotA interacts with DNA within the transcription complex. Conclusion: The unusual “double-wing” motif in MotACTD sits within the DNA major groove. Significance: FeBABE analyses together with structures can be used to determine protein-DNA architecture within multiprotein complexes. Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ70 subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ70 and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotANTD, MotACTD, and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual “double wing” motif present within MotACTD resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses.

Bacteriophage T4 MotA Activator and the β-Flap Tip of RNA Polymerase Target the Same Set of σ70 Carboxyl-terminal Residues

Background: Transcriptional activators interact with RNA polymerase to redefine gene expression. Results: A phage activator engages a region of the specificity factor of E. coli RNA polymerase, which is normally bound by another portion of RNA polymerase. Conclusion: Using an activator/co-activator system, the phage hijacks the host RNA polymerase. Significance: Small transcriptional factors acting on defined local regions of RNA polymerase can fundamentally change gene expression. Sigma factors, the specificity subunits of RNA polymerase, are involved in interactions with promoter DNA, the core subunits of RNA polymerase, and transcription factors. The bacteriophage T4-encoded activator, MotA, is one such factor, which engages the C terminus of the Escherichia coli housekeeping sigma factor, σ70. MotA functions in concert with a phage-encoded co-activator, AsiA, as a molecular switch. This process, termed sigma appropriation, inhibits host transcription while activating transcription from a class of phage promoters. Previous work has demonstrated that MotA contacts the C terminus of σ70, H5, a region that is normally bound within RNA polymerase by its interaction with the β-flap tip. To identify the specific σ70 residues responsible for interacting with MotA and the β-flap tip, we generated single substitutions throughout the C terminus of σ70. We find that MotA targets H5 residues that are normally engaged by the β-flap. In two-hybrid assays, the interaction of σ70 with either the β-flap tip or MotA is impaired by alanine substitutions at residues Leu-607, Arg-608, Phe-610, Leu-611, and Asp-613. Transcription assays identify Phe-610 and Leu-611 as the key residues for MotA/AsiA-dependent transcription. Phe-610 is a crucial residue in the H5/β-flap tip interaction using promoter clearance assays with RNA polymerase alone. Our results show how the actions of small transcriptional factors on a defined local region of RNA polymerase can fundamentally change the specificity of polymerase.

Determining the Architecture of a Protein–DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program

Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex.

The phage T4 MotA transcription factor contains a novel DNA binding motif that specifically recognizes modified DNA

Abstract During infection, bacteriophage T4 produces the MotA transcription factor that redirects the host RNA polymerase to the expression of T4 middle genes. The C-terminal ‘double-wing’ domain of MotA binds specifically to the MotA box motif of middle T4 promoters. We report the crystal structure of this complex, which reveals a new mode of protein-DNA interaction. The domain binds DNA mostly via interactions with the DNA backbone, but the binding is enhanced in the specific cognate structure by additional interactions with the MotA box motif in both the major and minor grooves. The linker connecting the two MotA domains plays a key role in stabilizing the complex via minor groove interactions. The structure is consistent with our previous model derived from chemical cleavage experiments using the entire transcription complex. α- and β-d-glucosyl-5-hydroxymethyl-deoxycytosine replace cytosine in T4 DNA, and docking simulations indicate that a cavity in the cognate structure can accommodate the modified cytosine. Binding studies confirm that the modification significantly enhances the binding affinity of MotA for the DNA. Consequently, our work reveals how a DNA modification can extend the uniqueness of small DNA motifs to facilitate the specificity of protein-DNA interactions.

The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness

The lytic bacteriophage T4 employs multiple phage-encoded early proteins to takeover the Escherichia coli host. However, the functions of many of these proteins are not known. In this study, we have characterized the T4 early gene motB, located in a dispensable region of the T4 genome. We show that heterologous production of MotB is highly toxic to E. coli, resulting in cell death or growth arrest depending on the strain and that the presence of motB increases T4 burst size 2-fold. Previous work suggested that motB affects middle gene expression, but our transcriptome analyses of T4 motBam vs. T4 wt infections reveal that only a few late genes are mildly impaired at 5 min post-infection, and expression of early and middle genes is unaffected. We find that MotB is a DNA-binding protein that binds both unmodified host and T4 modified [(glucosylated, hydroxymethylated-5 cytosine, (GHme-C)] DNA with no detectable sequence specificity. Interestingly, MotB copurifies with the host histone-like proteins, H-NS and StpA, either directly or through cobinding to DNA. We show that H-NS also binds modified T4 DNA and speculate that MotB may alter how H-NS interacts with T4 DNA, host DNA, or both, thereby improving the growth of the phage.