Lawrence P. McIntosh

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, ∼20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

An NMR approach to structural proteomics

The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins.

Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.

Ets‐1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets‐1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C‐terminus. The ETS domain folds into a helix‐turn‐helix motif on a four‐stranded anti‐parallel beta‐sheet scaffold. This structure places Ets‐1 in the winged helix‐turn‐helix (wHTH) family of DNA binding proteins and provides a model for interpreting the sequence conservation of the ETS domain and the specific interaction of Ets‐1 with DNA. The C‐terminal sequence of Ets‐1, which is mutated in the v‐Ets oncoprotein, forms an alpha‐helix that packs anti‐parallel to the N‐terminal helix of the ETS domain. In this position, the C‐terminal helix is poised to interact directly with an N‐terminal inhibitory region in Ets‐1 as well as the wHTH motif. This explains structurally the concerted role of residues flanking the ETS domain in the intramolecular inhibition of Ets‐1 DNA binding.

The Structural and Dynamic Basis of Ets-1 DNA Binding Autoinhibition

The transcription factor Ets-1 is regulated by the allosteric coupling of DNA binding with the unfolding of an α-helix (HI-1) within an autoinhibitory module. To understand the structural and dynamic basis for this autoinhibition, we have used NMR spectroscopy to characterize Ets-1ΔN301, a partially inhibited fragment of Ets-1. The NMR-derived Ets-1ΔN301 structure reveals that the autoinhibitory module is formed predominantly by the hydrophobic packing of helices from the N-terminal (HI-1, HI-2) and C-terminal (H4, H5) inhibitory sequences, along with H1 of the intervening DNA binding ETS domain. The intramolecular interactions made by HI-1 in Ets-1ΔN301 are similar to the intermolecular contacts observed in the crystal structure of an Ets-1ΔN300 dimer, confirming that the latter represents a domain-swapped species. 15N relaxation studies demonstrate that the backbone of the N-terminal inhibitory sequence is mobile on the nanosecond-picosecond and millisecond-microsecond time scales. Furthermore, hydrogen exchange measurements reveal that amide protons in helices HI-1 and HI-2 exchange with water at rates only ∼15- and ∼75-fold slower, respectively, than predicted for an unfolded polypeptide. These findings indicate that inhibitory helices are only marginally stable even in the absence of DNA. The energetic coupling of DNA binding with the facile unfolding of the labile HI-1 provides a mechanism for modulating Ets-1 DNA binding activity via protein partnerships, post-translational modifications, or mutations. Ets-1 autoinhibition illustrates how conformational equilibria within structural domains can regulate macromolecular interactions.

The solution structure and interactions of CheW from Thermotoga maritima.

Using protein from the hyperthermophile Thermotoga maritima, we have determined the solution structure of CheW, an essential component in the formation of the bacterial chemotaxis signaling complex. The overall fold is similar to the regulatory domain of the chemotaxis kinase CheA. In addition, interactions of CheW with CheA were monitored by nuclear magnetic resonance (NMR) techniques. The chemical shift perturbation data show the probable contacts that CheW makes with CheA. In combination with previous genetic data, the structure also suggests a possible binding site for the chemotaxis receptor. These results provide a structural basis for a model in which CheW acts as a molecular bridge between CheA and the cytoplasmic tails of the receptor.

Protein Dielectric Constants Determined from NMR Chemical Shift Perturbations

Understanding the connection between protein structure and function requires a quantitative understanding of electrostatic effects. Structure-based electrostatic calculations are essential for this purpose, but their use has been limited by a long-standing discussion on which value to use for the dielectric constants (ε(eff) and ε(p)) required in Coulombic and Poisson-Boltzmann models. The currently used values for ε(eff) and ε(p) are essentially empirical parameters calibrated against thermodynamic properties that are indirect measurements of protein electric fields. We determine optimal values for ε(eff) and ε(p) by measuring protein electric fields in solution using direct detection of NMR chemical shift perturbations (CSPs). We measured CSPs in 14 proteins to get a broad and general characterization of electric fields. Coulombs law reproduces the measured CSPs optimally with a protein dielectric constant (ε(eff)) from 3 to 13, with an optimal value across all proteins of 6.5. However, when the water-protein interface is treated with finite difference Poisson-Boltzmann calculations, the optimal protein dielectric constant (ε(p)) ranged from 2 to 5 with an optimum of 3. It is striking how similar this value is to the dielectric constant of 2-4 measured for protein powders and how different it is from the ε(p) of 6-20 used in models based on the Poisson-Boltzmann equation when calculating thermodynamic parameters. Because the value of ε(p) = 3 is obtained by analysis of NMR chemical shift perturbations instead of thermodynamic parameters such as pK(a) values, it is likely to describe only the electric field and thus represent a more general, intrinsic, and transferable ε(p) common to most folded proteins.

Remeasuring HEWL pKa values by NMR spectroscopy: Methods, analysis, accuracy, and implications for theoretical pKa calculations

Site‐specific pKa values measured by NMR spectroscopy provide essential information on protein electrostatics, the pH‐dependence of protein structure, dynamics and function, and constitute an important benchmark for protein pKa calculation algorithms. Titration curves can be measured by tracking the NMR chemical shifts of several reporter nuclei versus sample pH. However, careful analysis of these curves is needed to extract residue‐specific pKa values since pH‐dependent chemical shift changes can arise from many sources, including through‐bond inductive effects, through‐space electric field effects, and conformational changes. We have re‐measured titration curves for all carboxylates and His 15 in Hen Egg White Lysozyme (HEWL) by recording the pH‐dependent chemical shifts of all backbone amide nitrogens and protons, Asp/Glu side chain protons and carboxyl carbons, and imidazole protonated carbons and protons in this protein. We extracted pKa values from the resulting titration curves using standard fitting methods, and compared these values to each other, and with those measured previously by 1H NMR (Bartik et al., Biophys J 1994;66:1180–1184). This analysis gives insights into the true accuracy associated with experimentally measured pKa values. We find that apparent pKa values frequently differ by 0.5–1.0 units depending upon the nuclei monitored, and that larger differences occasionally can be observed. The variation in measured pKa values, which reflects the difficulty in fitting and assigning pH‐dependent chemical shifts to specific ionization equilibria, has significant implications for the experimental procedures used for measuring protein pKa values, for the benchmarking of protein pKa calculation algorithms, and for the understanding of protein electrostatics in general. Proteins 2011.

Arginine: Its pKa value revisited

Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pKa value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼12 often used in structure‐based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pKa value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pKa value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro‐environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group.

The crystal structure of MexR from Pseudomonas aeruginosa in complex with its antirepressor ArmR

The intrinsic antimicrobial resistance of the opportunistic human pathogen Pseudomonas aeruginosa is compounded in mutant strains that overexpress multidrug efflux pumps such as the prominent drug-proton antiporter, MexAB-OprM. The primary regulator of the mexAB-oprM operon is the MarR family repressor, MexR. An additional repressor, NalC, also regulates mexAB-oprM by controlling expression of ArmR, an antirepressor peptide that is hypothesized to prevent the binding of MexR to its cognate DNA operator via an allosteric protein–peptide interaction. To better understand how ArmR modulates MexR, we determined the MexR-binding region of ArmR as its C-terminal 25 residues and solved the crystal structure of MexR in a 2:1 complex with this ArmR fragment at 1.8 Å resolution. This structure reveals that the C-terminal residues of ArmR form a kinked α-helix, which occupies a pseudosymmetrical and largely hydrophobic binding cavity located at the centre of the MexR dimer. Although the ArmR-binding cavity partially overlaps with the small molecule effector-binding sites of other MarR family members, it possesses a larger and more complex binding surface to accommodate the greater size and specific physicochemical properties of a peptide effector. Comparison with the structure of apo-MexR reveals that ArmR stabilizes a dramatic conformational change that is incompatible with DNA-binding. Thus, this work defines the structural mechanism by which ArmR allosterically derepresses MexR-controlled gene expression in P. aeruginosa and reveals important insights into the regulation of multidrug resistance.

The highly conserved beta-hairpin of the paired DNA-binding domain is required for assembly of Pax-Ets ternary complexes.

ABSTRACT Pax family transcription factors bind DNA through the paired domain. This domain, which is comprised of two helix-turn-helix motifs and a β-hairpin structure, is a target of mutations in congenital disorders of mice and humans. Previously, we showed that Pax-5 (B-cell-specific activator protein) recruits proteins of the Ets proto-oncogene family to bind a composite DNA site that is essential for efficient transcription of the early-B-cell-specificmb-1 promoter. Here, evidence is provided for specific interactions between Ets-1 and the amino-terminal subdomains of Pax proteins. By tethering deletion fragments of Pax-5 to a heterologous DNA-binding domain, we show that 73 amino acids (amino acids 12 to 84) of its amino-terminal subdomain can recruit the ETS domain of Ets-1 to bind the composite site. Furthermore, an amino acid (Gln22) within the highly conserved β-hairpin motif of Pax-5 is essential for efficient recruitment of Ets-1. The ability to recruit Ets proteins to bind DNA is a shared property of Pax proteins, as demonstrated by cooperative DNA binding of Ets-1 with sequences derived from the paired domains of Pax-2 and Pax-3. The strict conservation of sequences required for recruitment of Ets proteins suggests that Pax-Ets interactions are important for regulating transcription in diverse tissues during cellular differentiation.