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B2. Lysozyme - Biology

B2.  Lysozyme - Biology


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The lysozyme enzyme, found in cells and secretions of vertebrates but also in viruses which infect bacteria, cleaves peptidoglycan GlcNAc (β 1->4) MurNAc repeat linkages (NAG-NAM) in the cell walls of bacteria and the GlcNAc (β 1->4) GlcNAc (poly-NAG) in chitin, found in the cells walls of certain fungi. Since these polymers are hydrophilic, the active site of the enzyme would be expected to contain a solvent-accessible channel into which the polymer could bind. The crystal structures of lysozyme and complexes of lysozyme and NAG have been solved to high resolution. The inhibitors and substrates form strong H bonds and some hydrophobic interactions with the enzyme cleft. Kinetic studies using (NAG)n polymers show a sharp increase in kcat as n increases from 4 to 5. The kcat for NAG6 and (NAG-NAM)3 are similar. Models studies have shown that for catalysis to occur, (NAG-NAM)3 binds to the active site with each sugar in the chair conformation except the fourth which is distorted to a half chair form, which labilizes the glycosidic link between the 4th and 5th sugars. Additional studies show that if the sugars that fit into the binding site are labeled A-F, then because of the bulky lactyl substituent on the NAM, residues C and E can not be NAM, which suggests that B, D and F must be NAM residues. Cleavage occurs between residues D and E.

A review of the chemistry of glycosidic bond (an acetal) formation and cleavage shows the acetal cleavage is catalyzed by acids and proceeds by way of an oxonium ion which exists in resonance form as a carbocation.

Catalysis by the enzyme involves Glu 35 and Asp 52 which are in the active site. Asp 52 is surrounded by polar groups but Glu 35 is in a hydrophobic environment. This should increase the apparent pKa of Glu 35, making it less likely to donate a proton and acquire a negative charge at low pH values, making it a better general acid at higher pH values. The general mechanism appears to involve:

  • binding of a hexasaccharide unit of the peptidoglycan with concomitant distortion of the D NAM.
  • protonation of the sessile acetal O by the general acid Glu 35 (with the elevated pKa), which facilitates cleavage of the glycosidic link and formation of the resonant stabilized oxonium ion.
  • Asp 52 stabilizes the positive oxonium through electrostatic catalysis. The distorted half-chair form of the D NAM stabilizes the oxonium which requires co-planarity of the substituents attached to the sp2 hybridized carbon of the carbocation resonant form (much like we saw with the planar peptide bond).
  • water attacks the stablized carbocation, forming the hemiacetal with release of the extra proton from water to the deprotonated Glu 35 reforming the general acid catalysis.

Binding and distortion of the D substituent of the substrate (to the half chair form as shown above) occurs before catalysis. Since this distortion helps stabilize the oxonium ion intermediate, it presumably stabilizes the transition state as well. Hence this enzyme appears to bind the transition state more tightly than the free, undistorted substrate, which is yet another method of catalysis.

pH studies show that side chains with pKa's of 3.5 and 6.3 are required for activity. These presumably correspond to Asp 52 and Glu 35, respectively. If the carboxy groups of lysozyme are chemically modified in the presence of a competitive inhibitor of the enzyme, the only protected carboxy groups are Asp 52 and Glu 35.

In an alternative mechanism, Asp 52 acts as a nucleophilic catalysis and forms a covalent bond with NAM, expelling a NAG leaving group with Glu 35 acting as a general acid. This alternative mechanism also is consistent with other β-glycosidic bond cleavage enzyme. Substrate distortion is also important in this alternative mechanism.

Figure: alternative mechanism


MINIMAL METHODS OF PROCESSING | Potential Use of Phages and/or Lysins

Juan Jofre , Maite Muniesa , in Encyclopedia of Food Microbiology , 1999

Lysins

Phage lysins , or endolysins, are enzymes which hydrolyse cell walls. They are synthesized during late gene expression in the lytic cycle of multiplication of most phages, thereby enabling the release of progeny phages.

The mechanisms of lysis are not the same in the case of all phages, and may even differ in the same bacterial host, depending on the phage. There are at least two different mechanisms. Some small phages, exemplified by the Escherichia coli phages ϕX174 and MS2, have developed a single gene for a lysin which cannot degrade murein (peptidoglycan). These phages simply cause cells to empty, leaving non-refractile, rod-shaped cell ghosts. However, the great majority of known phages has a more complex mechanism of lysis, involving two different kinds of enzymes, known as lysins and holins. Only the joint activity of the two enzymes leads to lysis of the host cell. Lysins, also known as endolysins, are highly efficient and specific peptidoglycan-hydrolysing enzymes, which are expressed as soluble cytoplasmic proteins. However lysins can only reach their peptidoglycan substrate in the cell wall through the action of a second group of phage-encoded proteins, known as holins. These produce holes in the cytoplasmic membrane, through which the lysin molecules move into the periplasm, where they come into contact with the peptidoglycan. This is the dominant strategy for lysis by phages of both Gram-positive bacteria (e.g. Lactococcus, Streptococcus, Listeria and Bacillus) and Gram- negative bacteria (e.g. E. coli, Salmonella and Haemophilus).

Phage-encoded endolysins can be of any one of several unrelated types of enzymes (e.g. lysozyme, amidase, transglycosylase). These attack either glycosidic bonds (lysozyme and transglycosylase) or peptide bonds (amidase), which in combination confer mechanical rigidity on peptidoglycan.

The lysins of the phages of Gram-positive bacteria were recognized early on as being strongly active against the cell walls of their host bacteria when added exogenously, i.e. the cells can be lysed from the outside by the specific lysin. In Gram-negative bacteria, the situation seems more complex. When these bacteria are infected with phages at a high multiplicity of infection, i.e. with many phages per host cell, the bacteria lyse before phage replication. This phenomenon is known as ‘lysis from without’, and is due partially to the phage lysins. However, at least one other molecule is needed, probably because the outer membrane of the bacterium hinders contact between the lysins and their peptidoglycan substrate. Thus it appears that independently, only the lysins of Gram-positive bacteria have clear exogenous activity.

Even when applied exogenously, lysins retain a certain degree of specificity, the causes of which are not yet well-understood. For example, when the lysins of listeriophages are applied exogenously they induce rapid lysis of Listeria strains from all species, but generally do not affect other bacteria. Even the lysins coded by phages with limited host ranges are exogenously active against all listerial cell walls, regardless of serovars and species. However, they do not affect other Gram-positive bacteria with the same peptidoglycan type (A1γ-variation of directly cross- linked meso-diaminopimelic acid peptidoglycan). See Loessner et al (1995) Molecular Biology 16(6): 1231–1241. The molecular basis of this substrate specificity remains to be elucidated. A similar pattern can be observed in the case of phages infecting a number of different lactic acid bacteria. Certain lysins, e.g. some pneumococcal lysins, may have a broader substrate specificity than others, and even have some activity in taxonomically unrelated bacteria. However, the specificity shown by the lysins of the phages of lactic acid bacteria and listeriophages is a general phenomenon, common to the lysins of the phages of Gram-positive bacteria. Thus phage lysins can be described as very specific compared to other antimicrobial agents, although less specific than phages. The differences in the structure of the cells walls of Gram-positive and Gram-negative bacteria suggest that the exogenous activity of lysins will be more efficient in Gram-positive than in Gram-negative bacteria, in which the outer membrane is likely to somehow obstruct contact between the phage lysins and their peptidoglycan substrate.


B2. Lysozyme - Biology

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.243 
  • R-Value Work: 0.199 
  • R-Value Observed: 0.202 

wwPDB Validation   3D Report Full Report

In meso in situ serial X-ray crystallography of soluble and membrane proteins.

(2015) Acta Crystallogr D Biol Crystallogr 71: 1238-1256

  • PubMed: 26057665  Search on PubMedSearch on PubMed Central
  • DOI: 10.1107/S1399004715005210
  • Primary Citation of Related Structures:  
    4XJB, 4XJD, 4XJF, 4XJG, 4XJH, 4XJI, 4XNI, 4XNJ, 4XNK, 4XNL
  • PubMed Abstract: 

The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method .

The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the β2-adrenoreceptor-Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.

Organizational Affiliation

Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry and Immunology, Trinity College, Dublin, Ireland.


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Science

Vol 318, Issue 5854
23 November 2007

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By Vadim Cherezov , Daniel M. Rosenbaum , Michael A. Hanson , Søren G. F. Rasmussen , Foon Sun Thian , Tong Sun Kobilka , Hee-Jung Choi , Peter Kuhn , William I. Weis , Brian K. Kobilka , Raymond C. Stevens

Science 23 Nov 2007 : 1258-1265


Acknowledgements

We thank R. Lee and S. Murphy for their contributions working with GT enzymes, L. Yates for helpful discussions with glyco-recoding, D. Mills for helpful discussions regarding O-OSTs, M. Paszek, J. Hershewe, K. Warfel, J. Stark, and M. Jewett for helpful discussions and provision of reagents, M. Li for technical advice and J. Wilson, J. Brooks and J. Merritt for help with vector design and yeast-based recombineering. We are also grateful to R. Bhawal and S. Zhang of the Proteomics and Metabolomics Core Facility in the Cornell Institute of Biotechnology for assistance with LC-MS. This work was supported by the Defense Threat Reduction Agency (GRANT11631647 to M.P.D.), National Science Foundation (grant no. CBET-1605242 to M.P.D.) and National Institutes of Health (grant no. 1R01GM127578-01 to M.P.D.). Glycomics analysis was supported in part by the National Institutes of Health (grant no. 1S10OD018530 to P.A.). The work was also supported by seed project funding (to M.P.D.) through the National Institutes of Health-funded Cornell Center on the Physics of Cancer Metabolism (supporting grant no. 1U54CA210184-01). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. T.J. was supported by a Royal Thai Government Fellowship and also a Cornell Fleming Graduate Scholarship. E.C.C. was supported by a National Institutes of Health Chemical-Biology Interface (CBI) training fellowship (supporting grant no. T32GM008500).


Supplementary Material

We thank Drs. Deanna Nguyen, Katsunori Shirane, and Kiyotaka Nagahama for helpful discussion, and Ms. Ichiko Kata for technical assistance.

This study was supported mainly by National Institutes of Health grant DK64351 (A. Mizoguchi) and partially by the Eli and Edythe L. Broad Foundation (A. Mizoguchi), DK47677 (A.K. Bhan), and DK64289 (E. Mizoguchi) and Center for the Study of Inflammatory Bowel Disease at Massachussetts General Hospital.

The authors have no conflicting financial interests.


Phage Lysis: Multiple Genes for Multiple Barriers

Jesse Cahill , Ry Young , in Advances in Virus Research , 2019

3.1 Canonical Endolysins

For canonical holin- endolysin lysis, a key feature is that the nature of the micron-scale hole makes the molecular details of the endolysin irrelevant. This notion is reinforced by comparing the lysis cassettes of lambda and P22, two of the best-studied paradigm phages, in which the holin genes are

95% identical but the endolysins are completely different enzymes: lambda R is a transglycosylase whereas P22 gp14 is a true lysozyme ( Bienkowska-Szewczyk and Taylor, 1980 Weaver et al., 1985 ). However, the ability of the diverse phage holins and endolysins to cross-complement has only been studied in bulk liquid culture. Significant differences might be revealed in single cell experiments with high-resolution phase contrast and fluorescence microscopy. For example, holins always have a small cytoplasmic domain, usually at the C-terminus and usually highly hydrophilic with basic character. It is possible that the endolysin is not distributed uniformly within the cytoplasm weak interactions with the holin may bias its localization to the vicinity of the rafts and thus potentiate more rapid attack on the PG after hole formation. It should be noted that canonical endolysins are usually produced in great excess, as is evident from the fact that double nonsense mutants are needed to obtain a clean lysis-defective phenotype for the lambda R endolysin gene (Ry Young, Unpublished results). Phage endolysins in general have been recently been the subject of heightened interest and investigation as novel bacteriocides, or “enzybiotics” ( Fischetti, 2010 ). This exciting development is based on the fact that most phages of Gram-positive hosts have endolysins that have separate cell wall binding (CWB) and enzymatic domains, usually N- and C-terminal, respectively ( Fischetti, 2005 ). The CWB domain confers genus-specificity on the endolysin, an attractive feature for development as a useful antibiotic. Endolysins of nearly all phages with Gram-negative hosts have a single domain combining PG-binding and enzymatic activity ( Briers et al., 2007 ). This difference probably originates from the fact that in Gram-positive cells the PG is a thick, multilayer barrier PG exposed to the medium. Among other considerations, unrestrained release of the endolysin from a cell undergoing phage lysis might cause unacceptable collateral damage to nearby cells that are potential prey. There are interesting exceptions in the case of two giant phages of Pseudomonas, phiKZ and EL ( Briers et al., 2007 ), which have endolysins with an N-terminal CWB and C-terminal catalytic domain. Both enzymes were shown to have extraordinarily high enzymatic activity,

100-fold more than egg white lysozyme, in cell wall degradation assays. However, nothing is known about the lysis pathway in these enormous phages, and the genome complexity has so far precluded identification of other lysis genes, so the significance of this unusual modularity is unknown.


MARs and transcriptional regulation

The tethering of DNA to the nuclear matrix plays a vital role in transcription [9, 21, 22]. Using T-cell differentiation as a model we will describe how MARs facilitate transcription and reveal how they shape chromatin architecture to insulate chromatin domains from the effects of flanking chromatin.

Upon stimulation by antigen, naive CD4 helper T cells differentiate into effector Th1 and Th2 cells. In mice, Ifng (the gene for the cytokine interferon-γ) is silenced in naive T cells but transcribed in activated Th1 cells. The architecture of the Ifng locus has been analyzed in these two cell types by a combination of chromosome conformation capture and microarray technology [22]. In naive T cells Ifng was found to exist in a linear conformation, but in Th1 cells it is present in a chromatin loop, due to tethering of DNA to the nuclear matrix by MARs 7 kb upstream and 14 kb downstream of the locus. The absence of this selective DNA attachment to the nuclear matrix in naive T cells suggests that dynamic DNA anchors mediate the formation of the looped structure and the expression of the Ifng locus [22].

The molecular mechanisms by which MARs reorganize higher-order chromatin structure have been investigated in detail at the murine Th2 cytokine locus, which contains the cluster of coordinately regulated genes Il4, Il13 and Il5 in a region of about 120 kb [23]. These genes are expressed in Th2 cells but are silent in naive T cells. Following Th2 activation, expression of the nuclear matrix protein SATB1 is rapidly induced, and MARs within the locus mediate the formation of small loops by anchoring the loops onto a common protein core associated with SATB1 [12]. Down-regulation of SATB1 expression by RNA interference prevents both the formation of this looped structure and transcriptional activation of the locus [12]. In SATB1-null thymocytes (developing T cells) the expression of many genes is spatially and temporally misregulated, and T-cell development in SATB1-deficient mice is prematurely blocked. These results indicate that the binding of SATB1 at MARs regulates the expression of T-cell differentiation genes by reorganizing higher-order chromatin architecture [24, 25]. A similar MAR-mediated loop-formation mechanism regulates expression of the human β-globin gene cluster [26, 27].

Cai et al. [25] reported that SATB1 recruits several chromatin-remodeling enzymes at MARs to activate or repress the expression of nearby sequences. Other studies have shown that MARs interact dynamically with basal components of the transcription machinery and with splicing factors [28, 29]. In eukaryotic cells, mRNA synthesis is concentrated at discrete transcription 'factories' or foci within the nucleus, which contain RNA polymerases, RNA transcripts, transcription factors and mRNA-processing factors [30]. The retention of RNA polymerase II and general transcription factors in nuclei after extraction of soluble proteins and nuclease digestion suggests that transcription factories are assembled onto the nuclear matrix [31, 32]. As MARs associate with components of transcription factories as well as the nuclear matrix, it is tempting to speculate that dynamic interactions between MARs and the matrix bring together proximal and distal regulatory sequences and localize them close to transcription factories, thus promoting efficient regulation of gene expression (Figure 1).

A simplified model depicting the function of matrix-attachment regions (MARs) in gene regulation. Activation of transcription is accompanied by the anchoring of MARs to the nuclear matrix. This results in the formation of an anchored chromatin loop that is insulated from the stimulatory or repressive effects of the flanking chromatin. The transcription machinery is assembled at the site of the MAR-nuclear matrix attachments. Interaction of MARs with the nuclear matrix brings together gene coding sequences, regulatory DNA elements and the transcription machinery, thus enabling specific genes to be coordinately regulated. At the end of S phase, the replication machinery is dismantled.

Many genes are known to be shielded by so-called 'insulator' elements from stimulatory or repressive effects attributable to the chromatin state and regulatory elements in flanking regions. MARs commonly map to sequences flanking genes, and co-localize with some of the most extensively analyzed insulator elements, including gypsy, a retrotransposon in Drosophila melanogaster, suggesting that MARs have an insulator function [33]. In Drosophila, the nuclear matrix protein Su(Hw) binds to gypsy, creating chromatin loops [34]. Certain mutations in Su(Hw) that disrupt the loop structures render the insulator non-functional [34, 35]. This suggests that the tethering of MARs to the nuclear matrix topologically constrains the DNA into looped structures, protecting the intervening DNA from the influence of cis-regulatory elements outside the loop. In vertebrates, CTCF, a ubiquitous nuclear matrix protein, binds to insulators and has also been shown to interact with MARs [36]. While the precise mechanisms of CTCF insulation remain unclear, the binding of CTCF to MARs might block interactions between promoters and unrelated enhancers and create looped structures that delimit different chromosomal domains [37]. Experiments in a wide variety of higher eukaryotes have shown that in stably transfected cells, MAR-containing transgenes were expressed at higher levels compared with transgenes lacking MARs, indicating that the MARs shield the transgenes from the effects of the neighboring host chromatin [38, 39].

Taken together, the experimental evidence described above supports the view that MARs function as landing platforms for a wide range of matrix proteins. Such interactions form complex higher-order nucleoprotein structures, which insulate chromatin domains and also control gene expression by forming bridges between components of the basal transcription machinery and distal and proximal regulatory elements. MARs can thus be defined as cis-acting elements constituting a critical layer of transcriptional regulation.


Contents

The ADRB2 gene is intronless. Different polymorphic forms, point mutations, and/or downregulation of this gene are associated with nocturnal asthma, obesity and type 2 diabetes. [7]

The 3D crystallographic structure (see figure and links to the right) of the β2-adrenergic receptor has been determined [8] [9] [10] by making a fusion protein with lysozyme to increase the hydrophilic surface area of the protein for crystal contacts. An alternative method, involving production of a fusion protein with an agonist, supported lipid-bilayer co-crystallization and generation of a 3.5 Å resolution structure. [11]

This receptor is directly associated with one of its ultimate effectors, the class C L-type calcium channel CaV1.2. This receptor-channel complex is coupled to the Gs G protein, which activates adenylyl cyclase, catalysing the formation of cyclic adenosine monophosphate (cAMP) which then activates protein kinase A, and counterbalancing phosphatase PP2A. Protein kinase A then goes on to phosphorylate (and thus inactivate) myosin light-chain kinase, which causes smooth muscle relaxation, accounting for the vasodilatory effects of beta 2 stimulation. The assembly of the signaling complex provides a mechanism that ensures specific and rapid signaling. A two-state biophysical and molecular model has been proposed to account for the pH and REDOX sensitivity of this and other GPCRs. [12]

Beta-2 adrenergic receptors have also been found to couple with Gi, possibly providing a mechanism by which response to ligand is highly localized within cells. In contrast, Beta-1 adrenergic receptors are coupled only to Gs, and stimulation of these results in a more diffuse cellular response. [13] This appears to be mediated by cAMP induced PKA phosphorylation of the receptor. [14] Interestingly, Beta-2 adrenergic receptor was observed to localize exclusively to the T-tubular network of adult cardiomyocytes, as opposed to Beta-1 adrenergic receptor, which is observed also on the outer plasma membrane of the cell [15]

Muscular system Edit

The β2 adrenoreceptor has been correlated with anabolic properties and muscular hypertrophy with usage of agents such as oral clenbuterol as well as intravenous albuterol, though oral albuterol did not generate the same impacts on muscle mass, suggesting that drugs with a short half-life do not maintain sufficient activation to achieve these effects. [16] [17] Long-acting β2 agonists such as clenbuterol (not used clinically in the United States) are frequently abused performance-enhancing drugs for their anabolic, lipolytic, and performance-enhancing effects. [18] As a result, most of these agents are banned by WADA (World Anti-Doping Agency), though some are permissible under a therapeutic use exemption and are typically monitored for usage in athletes. Clenbuterol remains banned not as a beta-agonist, but rather an anabolic agent.

Function Tissue Biological Role
Smooth muscle relaxation in: GI tract (decreases motility) Inhibition of digestion
Bronchi [19] Facilitation of respiration. Hence, beta-2 agonists can be useful in treating asthma.
Detrusor urinae muscle of bladder wall [20] [21] This effect is stronger than the alpha-1 receptor effect of contraction. Inhibition of need for micturition
Uterus Inhibition of labor
Seminal tract [22]
Increased perfusion and vasodilation Blood vessels and arteries to skeletal muscle including the smaller coronary arteries [23] and hepatic artery Facilitation of muscle contraction and motility
Increased mass and contraction speed Striated muscle [22]
Insulin and glucagon secretion Pancreas [24] Increased blood glucose and uptake by skeletal muscle
Glycogenolysis [22]
Tremor Motor nerve terminals. [22] Tremor is mediated by PKA mediated facilitation of presynaptic Ca 2+ influx leading to acetylcholine release.
Legend

Circulatory system Edit

  • Heart muscle contraction
  • Increase cardiac output (minor degree compared to β1).
    • Increases heart rate[19] in sinoatrial node (SA node) (chronotropic effect).
    • Increases atrialcardiac muscle contractility. (inotropic effect).
    • Increases contractility and automaticity[19] of ventricular cardiac muscle.

    Eye Edit

    In the normal eye, beta-2 stimulation by salbutamol increases intraocular pressure via net:

    • Increase in production of aqueous humour by the ciliary process,
    • Subsequent increased pressure-dependent uveoscleral outflow of humour, despite reduced drainage of humour via the Canal of Schlemm.

    In glaucoma, drainage is reduced (open-angle glaucoma) or blocked completely (closed-angle glaucoma). In such cases, beta-2 stimulation with its consequent increase in humour production is highly contra-indicated, and conversely, a topical beta-2 antagonist such as timolol may be employed.


    B2. Lysozyme - Biology

    Experimental Data Snapshot

    • Method: X-RAY DIFFRACTION
    • Resolution: 2.40 Å
    • R-Value Free: 0.232 
    • R-Value Work: 0.196 
    • R-Value Observed: 0.198 

    wwPDB Validation   3D Report Full Report

    High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.

    (2007) Science 318: 1258-1265

    • PubMed: 17962520  Search on PubMedSearch on PubMed Central
    • DOI: 10.1126/science.1150577
    • Primary Citation of Related Structures:  
      2RH1
    • PubMed Abstract: 

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2 .

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the beta2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.

    • GPCR Engineering Yields High-Resolution Structural Insights into beta2 Adrenergic Receptor Function.
      Rosenbaum, D.M., Cherezov, V., Hanson, M.A., Rasmussen, S.G.F., Thian, F.S., Kobilka, T.S., Choi, H.J., Yao, X.J., Weis, W.I., Stevens, R.C., Kobilka, B.K.
      () To be published --: --

    Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037, USA.


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