2.0 å structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability

AbstractBackground: Recent efforts to understand the basis of protein stability have focussed attention on comparative studies of proteins from hyperthermophilic and mesophilic organisms. Most work to date has been on either oligomeric enzymes or monomers comprising more than one domain. Such studies are hampered by the need to distinguish between stabilizing interactions acting between subunits or domains from those acting within domains. In order to simplify the search for determinants of protein stability we have chosen to study the monomeric enzyme indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus (sIGPS), which grows optimally at 90°C.Results The 2.0 å crystal structure of sIGPS was determined and compared with the known 2.0 å structure of the IGPS domain of the bifunctional enzyme from the mesophilic bacterium Escherichia coli (eIGPS). sIGPS and eIGPS have only 30% sequence identity, but share high structural similarity. Both are single-domain (β/α)8 barrel proteins, with one (eIGPS) or two (sIGPS) additional helices inserted before the first β strand. The thermostable sIGPS has many more salt bridges than eIGPS. Several salt bridges crosslink adjacent α helices or participate in triple or quadruple salt-bridge clusters. The number of helix capping, dipole stabilizing and hydrophobic interactions is also increased in sIGPS.Conclusion The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions. These include a larger number of salt bridges, stabilization of α helices and strengthening of both polypeptide chain termini and solvent-exposed loops

Structural features correlated with the extreme thermostability of 1[4Fe-4S] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima.

Understanding the molecular mechanisms behind extreme temperature stability is of relevance for the protein folding problem and for designing proteins for industrial and medical applications. A powerful approach for understanding the structural basis of thermostability is the comparison of high resolution structures of homologous proteins from mesophiles and thermophiles. The 1.75 A crystal structure of Thermotoga maritima 1[4Fe-4S] ferredoxin was compared with those of mesophilic ferredoxins. Detailed analysis of structural differences reveals that thermostability is achieved without large changes of the overall polypeptide chain folding. The most striking differences include the formation of additional hydrogen bonding networks involving both side-chain and main-chain atoms. These networks are mainly connecting turns and strongly fix the N-terminus to the central core of the protein, increasing the overall rigidity of Thermotoga maritima ferredoxin. Other possibly stabilizing factors are the shortening of a solvent exposed surface loop, the increased content of alanines in the second alpha-helix, and the replacement of three residues close to the iron-sulfur cluster, which are in energetically unfavourable conformations in other ferredoxins, by glycines

Small structural changes account for the high thermostability of 1[4Fe-4S] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima.

BACKGROUND: The characterization of the structural features that account for the high thermostability of some proteins is of great scientific and biotechnological interest. Proteins from hyperthermophilic organisms with optimum growth temperatures of 80 degrees C and higher generally show high intrinsic stabilities. The comparison of high resolution X-ray structures of these proteins with their counterparts from mesophilic organisms has therefore helped to identify potentially stabilizing forces in a number of cases. Small monomeric proteins which comprise only a single domain, such as ferredoxins, are especially suitable for such comparisons since the search for determinants of protein stability is considerably simplified. RESULTS: The 1.75 A crystal structure of the extremely thermostable 1[4Fe-4S] ferredoxin from Thermotoga maritima (FdTm) was determined and compared with other monocluster-containing ferredoxins with different degrees of thermostability. CONCLUSIONS: A comparison of the three-dimensional structure of FdTm with that of ferredoxins from mesophilic organisms suggests that the very high thermostability of FdTm is unexpectedly achieved without large changes of the overall protein structure. Instead, an increased number of potentially stabilizing features is observed in FdTm, compared with mesophilic ferredoxins. These include stabilization of alpha helices, replacement of residues in strained conformation by glycines, strong docking of the N-terminal methionine and an overall increase in the number of hydrogen bonds. Most of these features stabilize several secondary structure elements and improve the overall rigidity of the polypeptide backbone. The decreased flexibility will certainly play a relevant role in shielding the iron-sulfur cluster against physiologically high temperatures and further improve the functional integrity of FdTm

2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability.

BACKGROUND: Recent efforts to understand the basis of protein stability have focused attention on comparative studies of proteins from hyperthermophilic and mesophilic organisms. Most work to date has been on either oligomeric enzymes or monomers comprising more than one domain. Such studies are hampered by the need to distinguish between stabilizing interactions acting between subunits or domains from those acting within domains. In order to simplify the search for determinants of protein stability we have chosen to study the monomeric enzyme indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus (sIGPS), which grows optimally at 90 degrees C. RESULTS: The 2.0 A crystal structure of sIGPS was determined and compared with the known 2.0 A structure of the IGPS domain of the bifunctional enzyme from the mesophilic bacterium Escherichia coli (eIGPS). sIGPS and eIGPS have only 30% sequence identity, but share high structural similarity. Both are single-domain (beta/alpha)8 barrel proteins, with one (eIGPS) or two (sIGPS) additional helices inserted before the first beta strand. The thermostable sIGPS has many more salt bridges than eIGPS. Several salt bridges crosslink adjacent alpha helices or participate in triple or quadruple salt-bridge clusters. The number of helix capping, dipole stabilizing and hydrophobic interactions is also increased in sIGPS. CONCLUSIONS: The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions. These include a larger number of salt bridges, stabilization of alpha helices and strengthening of both polypeptide chain termini and solvent-exposed loops

An NMR-derived model for the solution structure of oxidized Thermotoga maritima 1[Fe4-S4] ferredoxin.

The solution structure of the 60-residue 1[Fe4-S4] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima was determined based on 683 distance and 35 dihedral angle restraints that were obtained from NMR data. In addition, data known from crystallographic studies of ferredoxins was used for modeling of the iron-sulfur cluster and its environment. The protein shows a globular fold very similar to the fold of the related 1[Fe4-S4] ferredoxins from Desulfovibrio gigas and Desulfovibrio africanus, and elements of regular secondary structure similar to those in other ferredoxins were found in the T. maritima protein. In particular, the T. maritima protein displayed a beta-sheet structure made up of strands located at the very NH(2) and COOH termini of the protein, and an internal alpha-helix. The internal beta-sheet observed in the D. gigas and D. africanus ferredoxins could not be confirmed in T. maritima ferredoxin and is thus suggested to be only weakly present or even absent in this protein. This result suggests that thermostability in ferredoxins is not necessarily correlated with the content of stable elements of regular secondary structure

Role of the N-terminal extension of the (betaalpha)8-barrel enzyme indole-3-glycerol phosphate synthase for its fold, stability, and catalytic activity.

Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fifth step in the biosynthesis of tryptophan. It belongs to the large and versatile family of (betaalpha)(8)-barrel enzymes but has an unusual N-terminal extension of about 40 residues. Limited proteolysis with trypsin of IGPS from both Sulfolobus solfataricus (sIGPS) and Thermotoga maritima (tIGPS) removes about 25 N-terminal residues and one of the two extra helices contained therein. To assess the role of the extension, the N-terminally truncated variants sIGPSDelta(1-26) and tIGPSDelta(1-25) were produced recombinantly in Escherichia coli, purified, and characterized in comparison to the wild-type enzymes. Both sIGPSDelta(1-26) and tIGPSDelta(1-25) have unchanged oligomerization states and turnover numbers. In contrast, their Michaelis constants for the substrate 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate are increased, and their resistance toward unfolding induced by heat and guanidinium chloride is decreased. sIGPSDelta(1-26) was crystallized, and its X-ray structure was solved at 2.8 A resolution. The comparison with the known structure of sIGPS reveals small differences that account for its reduced substrate affinity and protein stability. The structure of the core of sIGPSDelta(1-26) is, however, unchanged compared to sIGPS, explaining its retained catalytic activity and consistent with the idea that it evolved from the same ancestor as the phosphoribosyl anthranilate isomerase and the alpha-subunit of tryptophan synthase. These (betaalpha)(8)-barrel enzymes catalyze the reactions preceding and following IGPS in tryptophan biosynthesis but lack an N-terminal extension

An Additional Region of Coactivator GRIP1 Required for

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A C-Terminal Inhibitory Domain Controls the Activity of p63 by an Intramolecular Mechanism

The human genome is far smaller than originally estimated, and one explanation is that alternative splicing creates greater proteomic complexity than a simple count of open reading frames would suggest. The p53 homologue p63, for example, is a tetrameric transcription factor implicated in epithelial development and expressed as at least six isoforms with widely differing transactivation potential. In particular, p63α isoforms contain a 27-kDa C-terminal region that drastically reduces their activity and is of clear biological importance, since patients with deletions in this C terminus have phenotypes very similar to patients with mutations in the DNA-binding domain. We have identified a novel domain within this C terminus that is necessary and sufficient for transcriptional inhibition and which acts by binding to a region in the N-terminal transactivation domain of p63 homologous to the MDM2 binding site in p53. Based on this mechanism, we provide a model that explains the transactivation potential of homo- and heterotetramers composed of different p63 isoforms and their effect on p53