Biomedical subjects

Zbigniew Dauter

Publications and source records attributed to Zbigniew Dauter.

46 records · Page 3Linked to original sources

PubMed2002

The impact of Glu-->Ala and Glu-->Asp mutations on the crystallization properties of RhoGDI: the structure of RhoGDI at 1.3 A resolution.

It is hypothesized that surface residues with high conformational entropy, specifically lysines and glutamates, impede protein crystallization. In a previous study using a model system of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI), it was shown that mutating Lys residues to Ala results in enhanced crystallizability, particularly when clusters of lysines are targeted. It was also shown that one of these mutants formed crystals that yielded diffraction to 2.0 A, a significant improvement on the wild-type protein crystals. In the current paper, an analysis of the impact of surface mutations replacing Glu residues with Ala or Asp on the stability and crystallization properties of RhoGDI is presented. The Glu-->Ala (Asp) mutants are generally more likely to produce crystals of the protein than the wild-type and in one case the resulting crystals yielded a diffraction pattern to 1.2 A resolution. This occurs in spite of the fact that mutating surface Glu residues almost invariably affects the protein's stability, as illustrated by the reduced deltaG between folded and unfolded forms measured by isothermal equilibrium denaturation. The present study strongly supports the notion that rational surface mutagenesis can be an effective tool in overcoming problems stemming from the protein's recalcitrance to crystallization and may also yield dramatic improvements in crystal quality.

Alanine↗

PubMed2002

The structures of Micrococcus lysodeikticus catalase, its ferryl intermediate (compound II) and NADPH complex.

The crystal structure of the bacterial catalase from Micrococcus lysodeikticus has been refined using the gene-derived sequence both at 0.88 A resolution using data recorded at 110 K and at 1.5 A resolution with room-temperature data. The atomic resolution structure has been refined with individual anisotropic atomic thermal parameters. This has revealed the geometry of the haem and surrounding protein, including many of the H atoms, with unprecedented accuracy and has characterized functionally important hydrogen-bond interactions in the active site. The positions of the H atoms are consistent with the enzymatic mechanism previously suggested for beef liver catalase. The structure reveals that a 25 A long channel leading to the haem is filled by partially occupied water molecules, suggesting an inherent facile access to the active site. In addition, the structures of the ferryl intermediate of the catalase, the so-called compound II, at 1.96 A resolution and the catalase complex with NADPH at 1.83 A resolution have been determined. Comparison of compound II and the resting state of the enzyme shows that the binding of the O atom to the iron (bond length 1.87 A) is associated with increased haem bending and is accompanied by a distal movement of the iron and the side chain of the proximal tyrosine. Finally, the structure of the NADPH complex shows that the cofactor is bound to the molecule in an equivalent position to that found in beef liver catalase, but that only the adenine part of NADPH is visible in the present structure.

Amino Acid Sequence↗

PubMed2002

One-and-a-half wavelength approach.

In many cases single-wavelength anomalous diffraction (SAD) phasing leads to a successful structure solution, but it is impossible to predict beforehand if single-wavelength data with a certain amount of anomalous signal resulting will be sufficient. It is therefore safer to continue collecting data at different wavelengths according to a MAD protocol, but to simultaneously attempt to phase the first data set by the SAD method. If this is successful, then further data collection can be abandoned. This '1.5-wavelength' approach may save a substantial amount of time and effort and diminishes the effects of crystal radiation damage. The principles of SAD phasing are illustrated using vector diagrams in the Argand plane.

Crystallography, X-Ray↗

PubMed2002

Adenovirus proteinase: crystallization and preliminary X-ray diffraction studies to atomic resolution.

Adenovirus proteinase (AVP) is required for the synthesis of infectious virus and is a target for antiviral therapy. The enzyme requires two viral cofactors for activation: pVIc, an 11-amino acid peptide, and the viral DNA. The structure of the enzyme in the absence of cofactors has not been observed. Single crystals of AVP were obtained via microseeding using the hanging-drop vapour-diffusion method with sodium acetate and sodium citrate as precipitants. At the National Synchrotron Light Source at Brookhaven National Laboratory, the native crystal diffracted to a resolution of 0.98 A and an isomorphous heavy-atom derivative diffracted to 1.9 A. Comparison of the structure of AVP with that of the AVP-pVIc complex should reveal the structural basis of activation of the enzyme by pVIc.

Adenoviridae↗

PubMed2002

Structural origins of the functional divergence of human insulin-like growth factor-I and insulin.

Human insulin-like growth factors I and II (hIGF-I, hIGF-II) are potent stimulators of cell and growth processes. They display high sequence similarity to both the A and B chains of insulin but contain an additional connecting C-domain, which reflects their secretion without specific packaging or precursor conversion. IGFs also have an extension at the C-terminus known as the D-domain. This paper describes four homologous hIGF-1 structures, obtained from crystals grown in the presence of the detergent SB12, which reveal additional detail in the C- and D-domains. Two different detergent binding modes observed in the crystals may reflect different hIGF-I biological properties such as the interaction with IGF binding proteins and self-aggregation. While the helical core of hIGF-I is very similar to that in insulin, there are distinct differences in the region of hIGF-I corresponding to the insulin B chain C-terminus, residues B25-B30. In hIGF-I, these residues (24-29) and the following C-domain form an extensive loop protruding 20 A from the core, which results in a substantially different conformation for the receptor binding epitope in hIGF-I compared to insulin. One notable feature of the structures presented here is demonstration of peptide-bond cleavage between Ser35 and Arg36 resulting in an apparent gap between residues 35 and 39. The equivalent region of proinsulin is involved in hormone processing demanding a reassessment of the structural integrity of hIGF-I in relation to its biological function.

Amino Acid Sequence↗

PubMed2002

Atomic resolution data reveal flexibility in the structure of RNase Sa.

Ribonuclease from Streptomyces aureofaciens, the bacterial source for the industrial production of chlorotetracycline, is a guanylate endoribonuclease (RNase Sa; EC 3.1.27.3) which hydrolyses the phosphodiester bonds of single-stranded RNA at the 3'-side of guanosine nucleotides with high specificity. The structure of the enzyme was previously refined at atomic resolution (1.2 A) using room-temperature data. Here, the RNase Sa structure refined against 1.0 A data collected at cryogenic temperature is reported. There are two surface loops in molecule A and one in molecule B for which two main-chain conformations are modelled: these loops contain active-site residues. The separation for most of the corresponding main-chain atoms in the two conformations is about 0.8 A, with a maximum of 2.5 A. The two regions of dual conformation represent the most important differences in comparison with the structure determined at room temperature, where the corresponding loops have one conformation only but the largest degree of anisotropy. The flexibility of the loops observed in the structure of RNase Sa is directly linked to the need for the active site to interact productively with substrates and/or inhibitors.

Catalytic Domain↗

PubMed2002

The crystal structure of the endothelial protein C receptor and a bound phospholipid.

The endothelial cell protein C receptor (EPCR) shares approximately 20% sequence identity with the major histocompatibility complex class 1/CD1 family of molecules, accelerates the thrombin-thrombomodulin-dependent generation of activated protein C, a natural anticoagulant, binds to activated neutrophils, and can undergo translocation from the plasma membrane to the nucleus. Blocking protein C/activated protein C binding to the receptor inhibits not only protein C activation but the ability of the host to respond appropriately to bacterial challenge, exacerbating both the coagulant and inflammatory responses. To understand how EPCR accomplishes these multiple tasks, we solved the crystal structure of EPCR alone and in complex with the phospholipid binding domain of protein C. The structures were strikingly similar to CD1d. A tightly bound phospholipid resides in the groove typically involved in antigen presentation. The protein C binding site is outside this conserved groove and is distal from the membrane-spanning domain. Extraction of the lipid resulted in loss of protein C binding, which could be restored by lipid reconstitution. CD1d augments the immune response by presenting glycolipid antigens. The EPCR structure is a model for how CD1d binds lipids and further suggests additional potential functions for EPCR in immune regulation, possibly including the anti-phospholipid syndrome.

Animals↗

PubMed2002

Jolly SAD.

Examples of phasing macromolecular crystal structures based on single-wavelength anomalous dispersion (SAD) show that this approach is more powerful and may have more general application in structural biology than was anticipated. Better data-collection facilities and cryogenic techniques, coupled with powerful programs for data processing, phasing, density modification and automatic model building, means that the SAD approach may gain wide popularity owing to its simplicity, less stringent wavelength requirements and faster data collection and phasing than the multi-wavelength (MAD) approach. It can be performed at any wavelength where anomalous scattering can be observed, in many cases using laboratory X-ray sources.

Aldose-Ketose Isomerases↗

PubMed2002

New approaches to high-throughput phasing.

Recent progress in macromolecular phasing, in part stimulated by the high-throughput structural biology initiatives, has made this crucial stage of the elucidation of crystal structures easier and more automatic. A quick soak in various salts leads to the rapid incorporation of the anomalously scattering ions, suitable for phasing by MAD (multiwavelength anomalous dispersion), SAD (single-wavelength anomalous dispersion) or MIR (multiple isomorphous replacement) methods. The availability of stable synchrotron beam lines equipped with elaborate hardware control and sophisticated data processing programs makes it possible to collect very accurate diffraction data and to solve structures from the very weak anomalous signal of such atoms as sulfur or phosphorus, inherently present in macromolecules. The current progress in phasing, coupled with the parallel advances in protein crystallization, diffraction data collection and so on, suggests that, in the near future, the process of macromolecular crystal structure elucidation may become fully automatic.

Algorithms↗

PubMed2002

Molecular basis of mitomycin C resistance in streptomyces: structure and function of the MRD protein.

Mitomycin C (MC) is a potent anticancer agent. Streptomyces lavendulae, which produces MC, protects itself from the lethal effects of the drug by expressing several resistance proteins. One of them (MRD) binds MC and functions as a drug exporter. We report the crystal structure of MRD and its complex with an MC metabolite, 1,2-cis-1-hydroxy-2,7-diaminomitosene, at 1.5 A resolution. The drug is sandwiched by pi-stacking interactions of His-38 and Trp-108. MRD is a dimer. The betaalphabetabetabeta fold of the MRD molecule is reminiscent of methylmalonyl-CoA epimerase, bleomycin resistance proteins, glyoxalase I, and extradiol dioxygenases. The location of the binding site is identical to the ones in evolutionarily related enzymes, suggesting that the protein may have been recruited from a different metabolic pathway.

Amino Acid Sequence↗