Although the cation–pi pair, formed between a side chain or substrate cation and the negative electrostatic potential of a pi system on the face of an aromatic ring, has been widely discussed and has ...been shown to be important in protein structure and protein–ligand interactions, there has been little discussion of the potential structural and functional importance in proteins of the related anion–aromatic pair (i.e., interaction of a negatively charged group with the positive electrostatic potential on the ring edge of an aromatic group). We posited, based on prior structural information, that anion–aromatic interactions between the anionic Asp general base and Phe54 and Phe116 might be used instead of a hydrogen-bond network to position the general base in the active site of ketosteroid isomerase from Comamonas testosteroni as there are no neighboring hydrogen-bonding groups. We have tested the role of the Phe residues using site-directed mutagenesis, double-mutant cycles, and high-resolution X-ray crystallography. These results indicate a catalytic role of these Phe residues. Extensive analysis of the Protein Data Bank provides strong support for a catalytic role of these and other Phe residues in providing anion–aromatic interactions that position anionic general bases within enzyme active sites. Our results further reveal a potential selective advantage of Phe in certain situations, relative to more traditional hydrogen-bonding groups, because it can simultaneously aid in the binding of hydrophobic substrates and positioning of a neighboring general base.
Summary This paper presents a meta-analysis of high-resolution human leukocyte antigen (HLA) allele frequency data describing 497 population samples. Most of the datasets were compiled from studies ...published in eight journals from 1990 to 2007; additional datasets came from the International Histocompatibility Workshops and from the AlleleFrequencies.net database. In all, these data represent approximately 66,800 individuals from throughout the world, providing an opportunity to observe trends that may not have been evident at the time the data were originally analyzed, especially with regard to the relative importance of balancing selection among the HLA loci. Population genetic measures of allele frequency distributions were summarized across populations by locus and geographic region. A role for balancing selection maintaining much of HLA variation was confirmed. Further, the breadth of this meta-analysis allowed the ranking of the HLA loci, with DQA1 and HLA-C showing the strongest balancing selection and DPB1 being compatible with neutrality. Comparisons of the allelic spectra reported by studies since 1990 indicate that most of the HLA alleles identified since 2000 are very-low-frequency alleles. The literature-based allele-count data, as well as maps summarizing the geographic distributions for each allele, are available online.
Higher throughput methods to mount and collect data from multiple small and radiation‐sensitive crystals are important to support challenging structural investigations using microfocus synchrotron ...beamlines. Furthermore, efficient sample‐delivery methods are essential to carry out productive femtosecond crystallography experiments at X‐ray free‐electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high‐density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer‐based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni‐puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room‐temperature data collection from multiple samples. New automated routines have been incorporated into the Blu‐Ice/DCSS experimental control system to support grids, including semi‐automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid‐handling robots, as well as incubation chambers, which support vapor‐diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid‐handling robots and incubation chambers are described. Crystals were screened at LCLS‐XPP and SSRL BL12‐2 at room temperature and cryogenic temperatures.
A high‐density sample mount has been developed for efficient goniometer‐based sample delivery at synchrotron and XFEL sources.
The carboxysome is a bacterial organelle that functions to enhance the efficiency of CO2 fixation by encapsulating the enzymes ribulose bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic ...anhydrase. The outer shell of the carboxysome is reminiscent of a viral capsid, being constructed from many copies of a few small proteins. Here we describe the structure of the shell protein CsoS1A from the chemoautotrophic bacterium Halothiobacillus neapolitanus. The CsoS1A protein forms hexameric units that pack tightly together to form a molecular layer, which is perforated by narrow pores. Sulfate ions, soaked into crystals of CsoS1A, are observed in the pores of the molecular layer, supporting the idea that the pores could be the conduit for negatively charged metabolites such as bicarbonate, which must cross the shell. The problem of diffusion across a semiporous protein shell is discussed, with the conclusion that the shell is sufficiently porous to allow adequate transport of small molecules. The molecular layer formed by CsoS1A is similar to the recently observed layers formed by cyanobacterial carboxysome shell proteins. This similarity supports the argument that the layers observed represent the natural structure of the facets of the carboxysome shell. Insights into carboxysome function are provided by comparisons of the carboxysome shell to viral capsids, and a comparison of its pores to the pores of transmembrane protein channels.
We compared the binding affinities of ground state analogues for bacterial ketosteroid isomerase (KSI) with a wild-type anionic Asp general base and with uncharged Asn and Ala in the general base ...position to provide a measure of potential ground state destabilization that could arise from the close juxtaposition of the anionic Asp and hydrophobic steroid in the reaction’s Michaelis complex. The analogue binding affinity increased ∼1 order of magnitude for the Asp38Asn mutation and ∼2 orders of magnitude for the Asp38Ala mutation, relative to the affinity with Asp38, for KSI from two sources. The increased level of binding suggests that the abutment of a charged general base and a hydrophobic steroid is modestly destabilizing, relative to a standard state in water, and that this destabilization is relieved in the transition state and intermediate in which the charge on the general base has been neutralized because of proton abstraction. Stronger binding also arose from mutation of Pro39, the residue adjacent to the Asp general base, consistent with an ability of the Asp general base to now reorient to avoid the destabilizing interaction. Consistent with this model, the Pro mutants reduced or eliminated the increased level of binding upon replacement of Asp38 with Asn or Ala. These results, supported by additional structural observations, suggest that ground state destabilization from the negatively charged Asp38 general base provides a modest contribution to KSI catalysis. They also provide a clear illustration of the well-recognized concept that enzymes evolve for catalytic function and not, in general, to maximize ground state binding. This ground state destabilization mechanism may be common to the many enzymes with anionic side chains that deprotonate carbon acids.
A library of 68 brominated fragments was screened against a new crystal form of inhibited HIV‐1 protease in order to probe surface sites in soaking experiments. Often, fragments are weak binders with ...partial occupancy, resulting in weak, difficult‐to‐fit electron density. The use of a brominated fragment library addresses this challenge, as bromine can be located unequivocally via anomalous scattering. Data collection was carried out in an automated fashion using AutoDrug at SSRL. Novel hits were identified in the known surface sites: 3‐bromo‐2,6‐dimethoxybenzoic acid (Br6) in the flap site and 1‐bromo‐2‐naphthoic acid (Br27) in the exosite, expanding the chemistry of known fragments for development of higher affinity potential allosteric inhibitors. At the same time, mapping the binding sites of a number of weaker binding Br‐fragments provides further insight into the nature of these surface pockets.
X‐marks the spot: Weak fragment binding at surface sites is more easily detected in X‐ray structures when a halogen atom in the fragment can be localized via an anomalous difference peak. A brominated fragment library elucidates known surface binding sites on HIV‐1 protease.
Within the idiosyncratic enzyme active-site environment, side chain and ligand pK a values can be profoundly perturbed relative to their values in aqueous solution. Whereas structural inspection of ...systems has often attributed perturbed pK a values to dominant contributions from placement near charged groups or within hydrophobic pockets, Tyr57 of a Pseudomonas putida ketosteroid isomerase (KSI) mutant, suggested to have a pK a perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semisynthesis, 13C NMR spectroscopy, absorbance spectroscopy, and X-ray crystallography, was used to unravel the basis for this perturbed Tyr pK a. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of positively charged residues near Tyr57 raises its pK a rather than lowers it; this effect, and part of the increase in the Tyr pK a from the introduction of nearby anionic groups, arises from accompanying active-site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water molecule that is part of a stabilizing hydrogen-bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen-bond donors, two Tyr side chains, and a water molecule positioned by other side chains and by a water-mediated hydrogen-bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural analysis can greatly facilitate our understanding of enzyme active-site energetics. The extensive data set provided may also be a valuable resource for those wishing to extensively test computational approaches for determining enzymatic pK a values and energetic effects.
Within the idiosyncratic enzyme active-site environment, side chain and ligand pKa values can be profoundly perturbed relative to their values in aqueous solution. Whereas structural inspection of ...systems has often attributed perturbed pKa values to dominant contributions from placement near charged groups or within hydrophobic pockets, Tyr57 of a Pseudomonas putida ketosteroid isomerase (KSI) mutant, suggested to have a pKa perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semisynthesis, (13)C NMR spectroscopy, absorbance spectroscopy, and X-ray crystallography, was used to unravel the basis for this perturbed Tyr pKa. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of positively charged residues near Tyr57 raises its pKa rather than lowers it; this effect, and part of the increase in the Tyr pKa from the introduction of nearby anionic groups, arises from accompanying active-site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water molecule that is part of a stabilizing hydrogen-bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen-bond donors, two Tyr side chains, and a water molecule positioned by other side chains and by a water-mediated hydrogen-bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural analysis can greatly facilitate our understanding of enzyme active-site energetics. The extensive data set provided may also be a valuable resource for those wishing to extensively test computational approaches for determining enzymatic pKa values and energetic effects.
Important three‐dimensional spatial features such as depth and surface concavity can be difficult to convey clearly in the context of two‐dimensional images. In the area of macromolecular ...visualization, the computer graphics technique of ray‐tracing can be helpful, but further techniques for emphasizing surface concavity can give clearer perceptions of depth. The notion of diffusion accessibility is well‐suited for emphasizing such features of macromolecular surfaces, but a method for calculating diffusion accessibility has not been made widely available. Here we make available a web‐based platform that performs the necessary calculation by solving the Laplace equation for steady state diffusion, and produces scripts for visualization that emphasize surface depth by coloring according to diffusion accessibility. The URL is http://services.mbi.ucla.edu/DiffAcc/.
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