Robert O. Fox

The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites

The mRNA of Escherichia coli yedU gene is induced 31‐fold upon heat shock. The 31‐kD YedU protein, also calls Hsp31, is highly conserved in several human pathogens and has chaperone activity. We solved the crystal structure of YedU at 2.2 Å resolution. YedU monomer has an α/β/α sandwich domain and a small α/β domain. YedU is a dimer in solution, and its crystal structure indicates that a significant amount of surface area is buried upon dimerization. There is an extended hydrophobic patch that crosses the dimer interface on the surface of the protein. This hydrophobic patch is likely the substrate‐binding site responsible for the chaperone activity. The structure also reveals a potential protease‐like catalytic triad composed of Cys184, His185, and Asp213, although no enzymatic activity could be identified. YedU coordinates a metal ion using His85, His122, and Glu90. This 2‐His‐1‐carboxylate motif is present in carboxypeptidase A (a zinc enzyme), and a number of dioxygenases and hydroxylases that utilize iron as a cofactor, suggesting another potential function for YedU.

Water cluster calibration reduces mass error in electrospray ionization mass spectrometry of proteins

Herein we report a novel calibration routine for use in positive ion mode electrospray ionization mass spectrometry (ESI-MS). Monoisotopic masses were calculated for a series of water clusters and used as calibration reference files (available at http://www.hbcg.utmb.edu/xray). The water cluster series contains singly charged peaks every 18 Da, which allows calibration curves to be precisely defined over a broad mass-to-charge ratio range. Water clusters, induced by a combination of high flow rate and high cone voltage, were used to accurately calibrate a quadrupole mass spectrometer from 100 to 1900 m/z. Calibration curves thus generated have many more data points and greatly reduced standard deviations compared to those obtained from myoglobin, sodium iodide, cesium iodide, or poly(ethylene glycol) based calibration standards. This calibration routine reduces the error in protein mass measurements by a factor of 3, from ±0.01% to ±0.0035% at the 95% confidence limit. The implications of this increased mass accuracy and wider calibrated mass-to-charge ratio scale for the study of protein sequence, structure, and folding by ESI-MS are discussed.

Exploring the impact of polyproline II (PII) conformational bias on the binding of peptides to the SEM‐5 SH3 domain

The left‐handed polyproline II helical structure (PII) is observed to be a dominant conformation in the disordered states of protein and small polypeptide chains, even when no prolines are present in the sequence. Recently, in work by Ferreon and Hilser, the energetics associated with Ala and Gly substitutions at a surface exposed proline site were determined calorimetrically by measuring the binding energetics of Sos peptide variants to the C‐terminal Src Homology 3 domain of SEM‐5. The results were interpreted as a significant conformational bias toward the bound conformation (i.e., PII), even when the ligand is unbound. That study was not able to determine, however, whether the conformational bias of the peptides could be explained in terms other than that of a PII preference. Here, we test, using a computer algorithm based on the hard sphere collision (HSC) model, the notion of whether a bias in the unbound states of the peptide ligands is specific for the PII conformation, or if a bias to any other region of (φ, ψ) space can also result in the same observed binding energetics. The results of these computer simulations indicate that, of the regions of (φ, ψ) modeled for bias in the small peptides, only the bias to the PII conformation, and at rates of bias similar to the experimentally observed rates, quantitatively reproduced the experimental binding energetics.

NMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognition.

Flaviviruses cause many human diseases, including dengue fever, yellow fever, West Nile viral encephalitis, and hemorrhagic fevers, and are transmitted to their vertebrate hosts by infected mosquitoes and ticks. Domain III of the envelope protein (E‐D3) is considered to be the primary viral determinant involved in the virus–host‐cell receptor interaction, and thus represents an excellent target for antiviral drug development. Langat (LGT) virus is a naturally attenuated BSL‐2 TBE virus and is a model for the pathogenic BSL‐3 and BSL‐4 viruses in the serogroup. We have determined the solution structure of LGT‐E‐D3 using heteronuclear NMR spectroscopy. The backbone dynamics of LGT‐E‐D3 have been investigated using 15N relaxation measurements. A detailed analysis of the solution structure and dynamics of LGT‐E‐D3 suggests potential residues that could form a surface for molecular recognition, and thereby represent a target site for antiviral therapeutics design.

Directed discovery of bivalent peptide ligands to an SH3 domain

The Caenorhabditis elegans SEM‐5 SH3 domains recognize proline‐rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline‐rich binding sequence, tethered by a glycine linker to a disulfide‐closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n‐Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP‐G4‐L) with 1000‐fold increased affinity for the SEM‐5 C‐terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n‐Src loops and parts of the β‐sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non‐PXXP peptide to the p67phox SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP‐G4‐L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.

Characterizing the Role of Ensemble Modulation in Mutation-Induced Changes in Binding Affinity

Protein conformational fluctuations are key contributors to biological function, mediating important processes such as enzyme catalysis, molecular recognition, and allosteric signaling. To better understand the role of conformational fluctuations in substrate/ligand recognition, we analyzed, experimentally and computationally, the binding reaction between an SH3 domain and the recognition peptide of its partner protein. The fluctuations in this SH3 domain were enumerated by using an algorithm based on the hard sphere collision model, and the binding energetics resulting from these fluctuations were calculated using a structure-based energy function parametrized to solvent accessible surface areas. Surprisingly, this simple model reproduced the effects of mutations on the experimentally determined SH3 binding energetics, within the uncertainties of the measurements, indicating that conformational fluctuations in SH3, and in particular the RT loop region, are structurally diverse and are well-approximated by the randomly configured states. The mutated positions in SH3 were distant to the binding site and involved Ala and Gly substitutions of solvent exposed positions in the RT loop. To characterize these fluctuations, we applied principal coordinate analysis to the computed ensembles, uncovering the principal modes of conformational variation. It is shown that the observed differences in binding affinity between each mutant, and thus the apparent coupling between the mutated sites, can be described in terms of the changes in these principal modes. These results indicate that dynamic loops in proteins can populate a broad conformational ensemble and that a quantitative understanding of molecular recognition requires consideration of the entire distribution of states.

The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A

Replacement of a cis‐proline by glycine at position 114 in ribonuclease A leads to a large decrease in thermal stability and simplifies the refolding kinetics. A crystallographic approach was used to determine whether the decrease in thermal stability results from the presence of a cis glycine peptide bond, or from a localized structural rearrangement caused by the isomerization of the mutated cis 114 peptide bond. The structure was solved at 2.0 Å resolution and refined to an R‐factor of 19.5% and an Rfree of 21.9%. The overall conformation of the protein was similar to that of wild‐type ribonuclease A; however, there was a large localized rearrangement of the mutated loop (residues 110–117—a 9.3 Å shift of the Cα atom of residue 114). The peptide bond before Gly114 is in the trans configuration. Interestingly, a large anomalous difference density was found near residue 114, and was attributed to a bound cesium ion present in the crystallization experiment. The trans isomeric configuration of the peptide bond in the folded state of this mutant is consistent with the refolding kinetics previously reported, and the associated protein conformational change provides an explanation for the decreased thermal stability.

Overexpression and functional characterization of the extracellular domain of the human α1 glycine receptor

A novel truncated form (residues 1-214, with a randomized C-terminal tail) of the ligand-binding extracellular domain (ECD) of the human alpha1 glycine receptor (GlyR), with amino acids from the corresponding sequence of an acetylcholine binding protein (AChBP) substituted for two relatively hydrophobic membrane-proximal loops, was overexpressed using a baculovirus expression system. The mutant GlyR ECD, named GlyBP, was present in both soluble and membrane-associated fractions after cell lysis, though only the latter appeared to be in a native-like conformation capable of binding strychnine, a GlyR specific antagonist. The membrane-associated GlyBP was solubilized, and detergent/lipid/protein micelles were affinity purified. After detergent removal, GlyBP may be isolated in either aqueous or vesicular form. Binding assays and spectroscopic studies using circular dichroism and FRET are consistent with both forms adopting equivalent native-like conformations. Thus, GlyBP may be isolated as a soluble or membrane-associated assembly that serves as a structural and functional homologue of the ECD of GlyR.

Crystallization and preliminary X-ray diffraction analysis of Langat virus envelope protein domain III

The putative receptor-binding domain (domain III) of the flavivirus Langat envelope glycoprotein has been crystallized using the hanging-drop vapor-diffusion method at 277 K. Two distinct crystal morphologies were observed to grow under the same conditions. The crystal forms both belong to a trigonal space group, P3(1)21 or P3(2)21, with unit-cell parameters a = 80.93, c = 132.1 A and a = 104.8, c = 219.5 A for forms I and II, respectively. Complete data sets to 2.9 and 3.35 A, respectively, have been collected at 100 K with Cu Kalpha X-rays from a rotating-anode generator.

Calibration of spiral-readout image-plate detectors

An improved method for intensity-uniformity calibration of diffraction data collected on spiral-readout image-plate (IP) systems is described. This technique is applicable to all types of spiral-readout IP detectors. The procedure utilizes an attenuated direct-beam scan of the IP to generate a radial-sensitivity calibration table. Exposure and scanning of the calibration frame are done on the same time scale as typical data collections, and require no additional equipment. Specific examples are presented for use with Mac Science DIP2000 systems. The new radial calibration is shown to reduce significantly structure-based R factors. The improved radial calibration is also shown to lower Rmerge when the IP is offset from the beam center. In addition to improving data quality and statistics, this method provides a quick and simple diagnostic tool to monitor changes in the sensitivity of the IP detector as a function of age.