ProFunc: a server for predicting protein function from 3D structure

Laskowski, Roman A.; Watson, James D.; Thornton, Janet M.

doi:10.1093/nar/gki414

Cited by 599 publications

(486 citation statements)

References 27 publications

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“…Multiple sequence alignments were constructed by using CLUSTALX (43), and sequence conservation was mapped onto the protein structure by using the ProFunc server (44).…”

Section: Methodsmentioning

confidence: 99%

Toward the structural genomics of complexes: Crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis

Strong

Sawaya

Wang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

686

661

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The developing science called structural genomics has focused to date mainly on high-throughput expression of individual proteins, followed by their purification and structure determination. In contrast, the term structural biology is used to denote the determination of structures, often complexes of several macromolecules, that illuminate aspects of biological function. Here we bridge structural genomics to structural biology with a procedure for determining protein complexes of previously unknown function from any organism with a sequenced genome. From computational genomic analysis, we identify functionally linked proteins and verify their interaction in vitro by coexpression͞copurification. We illustrate this procedure by the structural determination of a previously unknown complex between a PE and PPE protein from the Mycobacterium tuberculosis genome, members of protein families that constitute Ϸ10% of the coding capacity of this genome. The predicted complex was readily expressed, purified, and crystallized, although we had previously failed in expressing individual PE and PPE proteins on their own. The reason for the failure is clear from the structure, which shows that the PE and PPE proteins mate along an extended apolar interface to form a four-␣-helical bundle, where two of the ␣-helices are contributed by the PE protein and two by the PPE protein. Our entire procedure for the identification, characterization, and structural determination of protein complexes can be scaled to a genome-wide level.computational biology ͉ protein structure ͉ functional linkages

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“…Multiple sequence alignments were constructed by using CLUSTALX (43), and sequence conservation was mapped onto the protein structure by using the ProFunc server (44).…”

Section: Methodsmentioning

confidence: 99%

Toward the structural genomics of complexes: Crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis

Strong

Sawaya

Wang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

686

661

View full text Add to dashboard Cite

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“…The highest rate of identity among the regulatory subunits (;30%) is observed for the N-terminal regions that contain ACT domains, which play a role in the activation of CSU. Although the sequence identity is low, a ProFunc (Laskowski et al 2005) search with the SSM program reveals that many proteins, not just those involved in amino acid metabolism, share a similar fold with the ACT domains from TM0549 and NE1324. Among these structurally similar proteins, those involved in amino acid metabolism are phosphoglycerate dehydrogenases (PDB codes: 1PSD, 1SC6, 1YBA, 1YGY), transcriptional regulators from the Lpr/AsnC family (PDB codes: 2CG4, 2CFX), and phenylalanine hydroxylase (PDB code: 1PHZ).…”

Section: Overall Structurementioning

confidence: 99%

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

et al. 2007

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Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 Å and 2.5 Å , respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H 2 O) 6 2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice.Keywords: acetohydroxyacid synthase; actolactate synthase; regulatory subunit; ACT domain; AHAS; protein refolding Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes two physiologically significant reactions in the synthesis of isoleucine, valine, and leucine. In the first reaction, which is an initial step in the synthesis of isoleucine, 2-aceto-2-hydroxy butyrate is produced by condensation of 2-ketobutyrate with pyruvate. In the second reaction, 2-acetolactate is synthesized from two pyruvate molecules, and this provides substrates for synthesis of valine and leucine (Umbarger 1978(Umbarger , 1987Chipman et al. 1998). This pathway of branched-chain amino acid biosynthesis is characteristic for bacteria, fungi, algae, and higher plants, but not for animals. Accordingly, AHAS inhibitors have been developed as herbicides which have found broad application (Short and Colborn 1999). The inhibitors of branched-chain amino acids synthesis are also being tested as potential antituberculosis agents (Grandoni et al. 1998;Zohar et al. 2003;Choi et al. 2005).Three different FAD-dependent AHAS isozymes (I, II, and III) are found in enterobacteria (e.g., Escherichia ), but most other organisms from the bacteria encode only a single AHAS enzyme, similar to isozyme III from E. coli. The acetohydroxyacid synthases are multimeric proteins, and most frequently their biological unit is composed of two catalytic subunits (CSU) and two small regulatory subunits (SSU). In the absence of the SSU subunit, the CSU dimer is unstable (Vyazmensky et al. 1996), suggesting that in the holoenzyme the AHAS dimer is stabilized by a SSU dimer. The large catalytic subunits also show very weak activity without their associated regulatory subunits (Weinstock et al. 1992;Pang and Duggleby 1999). The E. coli AHAS enzyme can be reconstituted with SSU subunits from other organisms (Porat et al. 2004), and the reconstituted enzymes form stable heterotetramers. The properly associated holoenzymes show full catalytic activity, and are also susceptible to valine inhibition.Previous mutagenesis studies (Mendel et al. 2001;Kaplun et al. 2006) revealed that the valine binding sites are located at the dimerizatio...

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“…Two programs, ProFunc (27) and ESpript (28), were used to consider both primary sequence and structural information for a diverse set of TAF5 sequences (see SI Fig. 7).…”

Section: Resultsmentioning

confidence: 99%

Structural analysis and dimerization potential of the human TAF5 subunit of TFIID

Bhattacharya

Takada

Jacobson

2007

Proc. Natl. Acad. Sci. U.S.A.

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TFIID is an essential factor required for RNA polymerase II transcription but remains poorly understood because of its intrinsic complexity. Human TAF5, a 100-kDa subunit of general transcription factor TFIID, is an essential gene and plays a critical role in assembling the 1.2 MDa TFIID complex. We report here a structural analysis of the TAF5 protein. Our structure at 2.2-Å resolution of the TAF5-NTD2 domain reveals an ␣-helical domain with distant structural similarity to RNA polymerase II CTD interacting factors. The TAF5-NTD2 domain contains several conserved clefts likely to be critical for TFIID complex assembly. Our biochemical analysis of the human TAF5 protein demonstrates the ability of the N-terminal half of the TAF5 gene to form a flexible, extended dimer, a key property required for the assembly of the TFIID complex.inititation complex ͉ transcription ͉ x-ray crystallography ͉ protein-protein interaction T he general transcription factor TFIID plays a central role in the recognition of core promoter elements and is used for accurate transcription initiation by RNA Pol II for a large class of genes. Recent studies in yeast indicate that the majority of genes present are TFIID dependent (1). TFIID is a multiprotein complex composed of the TATA box-binding protein (TBP) and 14 other TBP-associated factors (TAFs) which have been highly conserved during eukaryotic evolution (2). TFIID is the only general transcription factor with specific TATA box binding activity and has been shown to initiate recruitment of the other general transcription factors (TFIIA, TFIIB, TFIIE, TFIIF, TFIIH) along with RNA Pol II into a functional preinitiation complex (PIC) that forms at the start site of Pol II genes.TAF subunits seem to serve multiple functions within TFIID holocomplex. For instance, hTAF6 and hTAF9 have been reported to interact with the downstream promoter element, whereas TAF1 and TAF2 have been shown to bind to the initiator. The specific contacts with the promoter DNA by TFIID support basal transcription from promoters containing these elements and have revealed the role of several TAFs on transcription (3-6). TAF1, the largest subunit of TFIID is known to harbor multiple enzymatic activities (7-9) and is also involved in chromatin transactions by using its double bromodomains to contact acetylated histone tails (10, 11). Furthermore, many of the TAFs have been shown to be involved in interactions with gene specific activators and other general transcription factors either to stabilize the preinitiation complex (12) or to induce structural changes in them (13). TAFs are not only restricted to the TFIID complex but also can be found in the yeast SAGA (Spt-Ada-GCN5-acetyltransferase complex), human STAGA (Spt3-TAF9-Ada-GCN5-acetyltransferase complex), PCAF (p300/CBP-associated factor), or TFTC (TBP-free TAFIIcontaining complex) (14-16). The TAFs within these multiprotein complexes are involved in extensive protein-protein interactions and various studies indicate the emerging role of TAFs as cofactors wit...

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ProFunc: a server for predicting protein function from 3D structure

Cited by 599 publications

References 27 publications

Toward the structural genomics of complexes: Crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis

Toward the structural genomics of complexes: Crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

Structural analysis and dimerization potential of the human TAF5 subunit of TFIID

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