High-pressure krypton gas and statistical heavy-atom refinement: A successful combination of tools for macromolecular structure determination

The noble gas krypton is shown to bind to crystallized proteins in a similar way to xenon [Schiltz, Prange & Fourme (1994). J. Appl. Cryst. 27, 950-960]. Preliminary tests show that the major krypton binding sites are essentially identical to those of xenon. Noticeable substitution is achieved only at substantially higher pressures (above 50 x 10(5) Pa). As is the case for xenon, the protein complexes with krypton are highly isomorphous with the native structure so that these complexes can be used for phase determination in protein crystallography. Krypton is not as heavy as xenon, but its K-absorption edge is situated at a wavelength (0.86 Angstrom) that is readily accessible on synchrotron radiation sources. As a test case, X-ray diffraction data at the high-energy side of the K edge were collected on a crystal of porcine pancreatic elastase (molecular weight of 25.9 kDa) put under a krypton gas pressure of 56 x 10(5) Pa. The occupancy of the single Kr atom is approximately 0.5, giving isomorphous and anomalous scattering strengths of 15.2 and 1.9 e, respectively. This derivative could be used successfully for phase determination with the SIRAS method (single isomorphous replacement with anomalous scattering). After phase improvement by solvent flattening, the resulting electron-density map is of exceptionally high quality, and has a correlation coefficient of 0.85 with a map calculated from the refined native structure. Careful data collection and processing, as well as the correct statistical treatment of isomorphous and anomalous signals have proven to be crucial in the determination of this electron-density map. Heavy-atom refinement and phasing were carried out with the program SHARP, which is a fully fledged implementation of the maximum-likelihood theory for heavy-atom refinement [Bricogne (1991). Crystallographic Computing 5, edited by D. Moras, A. D. Podjarny & J. C. Thierry, pp. 257-297. Oxford: Clarendon Press]. It is concluded that the use of xenon and krypton derivatives, when they can be obtained, associated with statistical heavy-atom refinement will allow one to overcome the two major limitations of the isomorphous replacement method i.e. non-isomorphism and the problem of optimal estimation of heavy-atom parameters

Protein crystallography at ultra-short wavelengths: Feasibility study of anomalous-dispersion experiments at the xenon K-edge

A protein crystallography experiment at the xenon K-edge (lambda = 0.358 Angstrom) has been successfully carried out at the materials science beamline (BL2/ID11) of the ESRF. The samples used in this methodological study were crystals of porcine pancreatic elastase, a 26 kDa protein of known structure. The diffraction data are of excellent quality. The combination of isomorphous replacement and anomalous dispersion of a single xenon heavy-atom derivative allowed accurate phase determination and the computation of a high-quality electron density map of the protein molecule. This is the first fully documented report on a complete protein crystallography experiment, from data collection up to phase determination and calculation of an electron density map, carried out with data obtained at ultra-short wavelengths. Experimental considerations as well as possible advantages and drawbacks of protein crystallography at very short and ultra-short wavelengths are discussed

Interferon-lambda is functionally an interferon but structurally related to the IL-10 family

Interferon-λ (IFN-λ) is an antiviral cytokine that signals through a distinct receptor complex, composed of the IFN-λR1 and interleukin-10R2 (IL-10R2) receptor chains. We have determined the crystal structure of human IFN-λ3 and characterized the interaction with its receptor complex through structure-based site-directed mutagenesis. The ability of IFN-λ3 mutants to signal was determined by measuring the antiviral activity and induced STAT2 phosphorylation. In conclusion, our data show that, although IFN-λ is functionally an interferon, it is clearly structurally related to members of the IL-10 family. In particular, we found an interesting similarity between IFN-λ and IL-22, and we suggest that IFN-λ and IL-22 possess parallel functions, protecting epithelial tissue against viral and bacterial infections, respectively

Repetitive architecture of the Haemophilus influenzae hia trimeric autotransporter

The Hia autotransporter of Haemophilus influenzae belongs to the trimeric autotransporter subfamily and mediates bacterial adherence to the respiratory epithelium. In this report, we show that the structure of Hia is characterized by a modular architecture containing repeats of structurally distinct domains. Comparison of the structures of HiaBD1 and HiaBD2 adhesive repeats and a nonadhesive repeat (a novel fold) shed light on the structural determinants of Hia adhesive function. Examination of the structure of an extended version of the Hia translocator domain revealed the structural transition between the C-terminal translocator domain and the N-terminal passenger domain, highlighting a highly intertwined domain that is ubiquitous among trimeric autotransporters. Overall, this study provides important insights into the mechanism of Hia adhesive activity and the overall structure of trimeric autotransporters

Crystal Structure of Leishmania major Oligopeptidase B Gives Insight into the Enzymatic Properties of a Trypanosomatid Virulence Factor*

Oligopeptidase B (OPB) is a serine peptidase with dibasic substrate specificity. It is found in bacteria, plants, and trypanosomatid pathogens, where it has been identified as a virulence factor and potential drug target. In this study we expressed active recombinant Leishmania major OPB and provide the first structure of an oligopeptidase B at high resolution. The crystallographic study reveals that OPB comprises two domains, a catalytic and a propeller domain, linked together by a hinge region. The structure has been determined in complex with the oligopeptide, protease-inhibitor antipain, giving detailed information on the enzyme active site and extended substrate binding pockets. It shows that Glu-621 plays a critical role in the S1 binding pocket and, along with Phe-603, is largely responsible for the enzyme substrate specificity in P1. In the S2 binding pocket, Tyr-499 was shown to be important for substrate stability. The structure also allowed an investigation into the function of residues highlighted in other studies including Glu-623, which was predicted to be involved in the S1 binding pocket but is found forming an inter-domain hydrogen bond. Additional important salt bridges/hydrogen bonds between the two domains were observed, highlighting the significance of the domain interface in OPB. This work provides a foundation for the study of the role of OPBs as virulence factors in trypanosomatids. It could facilitate the development of specific OPB inhibitors with therapeutic potential by exploiting its unique substrate recognition properties as well as providing a model for OPBs in general

Structure of the outer membrane translocator domain of the Haemophilus influenzae Hia trimeric autotransporter

Autotransporter proteins are defined by the ability to drive their own secretion across the bacterial outer membrane. The Hia autotransporter of Haemophilus influenzae belongs to the trimeric autotransporter subfamily and mediates bacterial adhesion to the respiratory epithelium. In this report, we present the crystal structure of the C-terminal end of Hia, corresponding to the entire Hia translocator domain and part of the passenger domain (residues 992–1098). This domain forms a β-barrel with 12 transmembrane β-strands, including four strands from each subunit. The β-barrel has a central channel of 1.8 nm in diameter that is traversed by three N-terminal α-helices, one from each subunit. Mutagenesis studies demonstrate that the transmembrane portion of the three α-helices and the loop region between the α-helices and the neighboring β-strands are essential for stability of the trimeric structure of the translocator domain, and that trimerization of the translocator domain is a prerequisite for translocator activity. Overall, this study provides important insights into the mechanism of translocation in trimeric autotransporters