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• W2010392743 abstract "Biotin protein ligase (BPL) catalyzes the biotinylation of the biotin carboxyl carrier protein (BCCP) only at a special lysine residue. Here we report the first structure of BPL·BCCP complex crystals, which are prepared using two BPL mutants: R48A and R48A/K111A. From a detailed structural characterization, it is likely that the mutants retain functionality as enzymes but have a reduced activity to produce the reaction intermediate biotinyl-5′-AMP. The observed biotin and partly disordered ATP in the mutant structures may act as a non-reactive analog of the substrates or biotinyl-5′-AMP, thereby providing the complex crystals. The four crystallographically independent BPL·BCCP complexes obtained can be classified structurally into three groups: the formation stages 1 and 2 with apo-BCCP and the product stage with biotinylated holo-BCCP. Residues responsible for the complex formation as well as for the biotinylation reaction have been identified. The C-terminal domain of BPL shows especially large conformational changes to accommodate BCCP, suggesting its functional importance. The formation stage 1 complex shows the closest distance between the carboxyl carbon of biotin and the special lysine of BCCP, suggesting its relevance to the unobserved reaction stage. Interestingly, bound ATP and biotin are also seen in the product stage, indicating that the substrates may be recruited into the product stage complex before the release of holo-BCCP, probably for the next reaction cycle. The existence of formation and product stages before and after the reaction stage would be favorable to ensure both the reaction efficiency and the extreme substrate specificity of the biotinylation reaction. Biotin protein ligase (BPL) catalyzes the biotinylation of the biotin carboxyl carrier protein (BCCP) only at a special lysine residue. Here we report the first structure of BPL·BCCP complex crystals, which are prepared using two BPL mutants: R48A and R48A/K111A. From a detailed structural characterization, it is likely that the mutants retain functionality as enzymes but have a reduced activity to produce the reaction intermediate biotinyl-5′-AMP. The observed biotin and partly disordered ATP in the mutant structures may act as a non-reactive analog of the substrates or biotinyl-5′-AMP, thereby providing the complex crystals. The four crystallographically independent BPL·BCCP complexes obtained can be classified structurally into three groups: the formation stages 1 and 2 with apo-BCCP and the product stage with biotinylated holo-BCCP. Residues responsible for the complex formation as well as for the biotinylation reaction have been identified. The C-terminal domain of BPL shows especially large conformational changes to accommodate BCCP, suggesting its functional importance. The formation stage 1 complex shows the closest distance between the carboxyl carbon of biotin and the special lysine of BCCP, suggesting its relevance to the unobserved reaction stage. Interestingly, bound ATP and biotin are also seen in the product stage, indicating that the substrates may be recruited into the product stage complex before the release of holo-BCCP, probably for the next reaction cycle. The existence of formation and product stages before and after the reaction stage would be favorable to ensure both the reaction efficiency and the extreme substrate specificity of the biotinylation reaction. Biotin-dependent carboxylases constitute a ubiquitous family of enzymes that catalyze the transfer of carbon dioxide between metabolites using the biotin moiety as a carboxyl carrier (1Knowles J.R. Annu. Rev. Biochem. 1989; 58: 195-221Crossref PubMed Scopus (355) Google Scholar, 2Chapman-Smith A. Cronan Jr., J.E. Trends Biochem. Sci. 1999; 24: 359-363Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The attachment of biotin to the biotin-dependent enzymes is catalyzed by the biotin protein ligase (BPL) 2The abbreviations used are: BPL, biotin protein ligase; BCCP, biotin carboxyl carrier protein; r.m.s.d., root mean square deviation. 2The abbreviations used are: BPL, biotin protein ligase; BCCP, biotin carboxyl carrier protein; r.m.s.d., root mean square deviation. in two steps (Reactions 1 and 2). Firstly, BPL activates biotin at the expense of ATP to the reaction intermediate biotinyl-5′-AMP in which the carboxyl group of inert biotin is activated by the addition of an adenylate group. Subsequently, the biotin moiety of biotinyl-5′-AMP is transferred to the ϵ-amino group of a specific lysine residue of the target protein (e.g. the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase (3Beckett D. Matthews B.W. Methods Enzymol. 1997; 279: 362-375Crossref PubMed Scopus (23) Google Scholar)). The biotinylated holo-BCCP subunit carries a covalently bound carboxyl unit between different active sites of the multienzyme biotin-dependent carboxylase complexes, which play essential roles in the fatty acid synthesis, the amino acid degradation, and the CO2 fixation (4Perham R.N. Annu. Rev. Biochem. 2000; 69: 961-1004Crossref PubMed Scopus (468) Google Scholar, 5Menendez C. Bauer Z. Huber H. Gad'on N. Stetter K.-O. Fuchs G. J. Bacteriol. 1999; 181: 1088-1098Crossref PubMed Google Scholar). In bacteria and eukaryotes, biotinylation is essential to initiate the first step of fatty acid biosynthesis, which is catalyzed by acetyl-CoA carboxylase. Because human biotin-dependent carboxylases are the direct targets for the development of anti-obesity and anti-diabetes agents, structural information on these enzymes is important for drug discovery research (6Tong L. Harwood Jr., H.J. J. Cell Biochem. 2006; 99: 1476-1488Crossref PubMed Scopus (162) Google Scholar). In archaea, the function of acetyl-CoA/propionyl-CoA carboxylases is different due to the absence of usual fatty acids in membranes: they act as CO2 fixation enzymes in the modified 3-hydroxypropionate cycle to assimilate CO2 into the cell (5Menendez C. Bauer Z. Huber H. Gad'on N. Stetter K.-O. Fuchs G. J. Bacteriol. 1999; 181: 1088-1098Crossref PubMed Google Scholar). In addition, the structural basis of biotinylation is important to develop useful applications in protein engineering, such as the high affinity biotin tagging for protein purification (7de Boer E. Rodriguez P. Bonte E. Krijgsveld J. Katsantoni E. Heck A. Grosveld F. Strouboulis J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7480-7485Crossref PubMed Scopus (344) Google Scholar) and the molecular imaging by quantum dots (8Howarth M. Takao K. Hayashi Y. Ting A.Y. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7583-7588Crossref PubMed Scopus (457) Google Scholar). The understanding of the first step of BPL reaction at the atomic level was reached based on the crystal structures of BPL from Pyrococcus horikoshii OT3 (PhBPL) in complex with various biological ligands including biotinyl-5′-AMP (9Bagautdinov B. Kuroishi C. Sugahara M. Kunishima N. J. Mol. Biol. 2005; 353: 322-333Crossref PubMed Scopus (49) Google Scholar) as well as on the crystal structure of BPL from Escherichia coli (EcBirA) in complex with the reaction intermediate analog biotinol-5′-AMP (10Wood Z.A. Weaver L.H. Brown P.H. Beckett D. Matthews B.W. J. Mol. Biol. 2006; 357: 509-523Crossref PubMed Scopus (69) Google Scholar). However, scientists have not yet been able to resolve the structural details in the second step of biotinylation reaction in which the activated biotin is transferred to the special lysine of BCCP. This exceptionally selective post-translational modification makes understanding how the proteins BPL and BCCP carry out the biotin transfer of particular interest. To elucidate the biotin transfer reaction, several biophysical or biochemical studies on the BPL·BCCP complex have been performed (e.g. an NMR study (11Reche P. Howard M.J. Broadhust R.W. Perham R.N. FEBS Lett. 2000; 479: 93-98Crossref PubMed Scopus (14) Google Scholar), mutagenesis studies (12Polyak S.W. Chapman-Smith A. Mulhern T.D. Cronan Jr., J.E. Wallace J.C. J. Biol. Chem. 2001; 276: 3037-3045Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 13Weaver L.H. Kwon K. Beckett D. Matthews B.W. Protein Sci. 2001; 10: 2618-2622Crossref PubMed Scopus (48) Google Scholar), and a chemical cross-linking study (14Clarke D.J. Coulson J. Baillie R. Campopiano D.J. Eur. J. Biochem. 2003; 270: 1277-1287Crossref PubMed Scopus (16) Google Scholar)). Despite these efforts, the structural mode of the biotin transfer reaction is not fully understood due to the absence of three-dimensional structures for the BPL·BCCP complex. To gain insight into a complex array of interactions between BPL and BCCP at biotinylation, we cocrystallized and determined the crystal structures of BPL·BCCP from P. horikoshii OT3. For the experiments, we used the biotinyl domain of PhBCCP, the C-terminal 73-residue fragment of the 149-residue long hypothetical methylmalonyl-CoA decarboxylase gamma chain (PhBCCPΔN76). Although the cocrystallization of the wild-type PhBPL with PhBCCPΔN76 was unsuccessful, using PhBPL mutants carrying a single mutation R48A (PhBPL*), and a double mutation R48A/K111A (PhBPL**), successfully yielded the complex crystals. Here we report the crystal structures of PhBPL*, PhBPL*·biotinyl-5′-AMP, PhBPL**, PhBPL**·biotin·adenosine, PhBPL**·biotinol-5′-AMP, PhBCCPΔN76, and the complexes PhBPL*·biotin·adenosine·PhBCCPΔN76 and PhBPL**·biotin·adenosine·holo-PhBCCPΔN76. The information obtained from these structures provides a good starting point to understand the structural basis of protein biotinylation. Protein Expression, Purification, Crystallization, and Data Collection—The expression and purification of PhBPL, PhBPL*, PhBPL**, and PhBCCPΔN76 were performed as described elsewhere (15Bagautdinov B. Kuroishi C. Sugahara M. Kunishima N. Acta Crystallogr. F. 2005; 61: 193-195Crossref Scopus (2) Google Scholar, 16Bagautdinov B. Matsuura Y. Bagautdinova S. Kunishima N. Acta Crystallogr. F. 2007; 63: 334-337Crossref Scopus (7) Google Scholar). Crystals of the ATP liganded form of wild-type PhBPL (PhBPL·ATP) were prepared by adding 5 mm ATP to the previously reported crystallization condition for the unliganded wild-type PhBPL (15Bagautdinov B. Kuroishi C. Sugahara M. Kunishima N. Acta Crystallogr. F. 2005; 61: 193-195Crossref Scopus (2) Google Scholar). Crystals of the liganded forms of PhBPL mutants were prepared using the wild-type crystallization condition except for adding ligands: 5 mm ATP and 5 mm biotin for PhBPL*·biotinyl-5′-AMP and PhBPL**·biotin·adenosine; 5 mm biotinol-5′-AMP for PhBPL**·biotinol-5′-AMP. Other forms of crystals were prepared as described elsewhere (16Bagautdinov B. Matsuura Y. Bagautdinova S. Kunishima N. Acta Crystallogr. F. 2007; 63: 334-337Crossref Scopus (7) Google Scholar). All data were collected at 100 K using synchrotron radiation on a Jupiter 210 charge-coupled device at the beamline BL26B1 of SPring-8, Japan (17Ueno G. Kanda H. Hirose R. Ida K. Kumasaka T. Yamamoto M. J. Struct. Funct. Genomics. 2006; 7: 15-22Crossref PubMed Scopus (89) Google Scholar). Diffraction data were processed and scaled with the HKL-2000 program suite (18Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38446) Google Scholar). Data collection statistics are summarized in tables (Table 1 and supplemental Tables S1 and S2).TABLE 1Crystallographic data collection and refinement statistics of PhBPL·PhBCCP complexesPhBPL*·biotin·adenosine·PhBCCP ΔN76PhBPL**·biotin·adenosine·holo-PhBCCP ΔN76Data collectionSpace groupP21P21Unit-cell parametersa, b, c (Å)69.57, 63.49, 74.7269.85, 63.12, 75.64β (°)93.795.9Resolution range (Å)50.0-2.70 (2.80-2.70)50.0-2.00 (2.07-2.00)No. of unique reflections15,564 (1378)42,152 (3935)Redundancy3.1 (2.8)3.2 (3.0)Completeness (%)95.1 (86.6)95.8 (90.0)<I/σ(I)>6.9 (2.2)9.0 (2.5)Rmerge (%)aRmerge = ∑h∑i|I(h)i - <I(h)>|/∑h∑i I(h)i, where I(h)i is the ith observation of the intensity of reflection h and <I(h)> is the mean value of all I(h)i.10.7 (32.0)8.7 (31.3)RefinementResolution (Å)38.9-2.7037.6-2.00Proteins atoms4,5954,646Ligand atoms7076Water oxygen atoms226409Rfactor (%)bRfactor = ∑|Fobs - Fcalc|/∑|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively.21.620.9Rfree (%)cRfree is the Rfactor for a subset of 5% of the reflections that were omitted from refinement.27.324.5Mean B-value (Å2)42.128.4Estimated coordinate error (Å)0.310.23r.m.s.d.Bond lengths (Å)0.0090.007Bond angles (°)1.41.2Ramachandran plotFavored (%)87.190.1Additional (%)12.59.7Generous (%)0.40.2PDB code2EJG2EJFa Rmerge = ∑h∑i|I(h)i - <I(h)>|/∑h∑i I(h)i, where I(h)i is the ith observation of the intensity of reflection h and <I(h)> is the mean value of all I(h)i.b Rfactor = ∑|Fobs - Fcalc|/∑|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively.c Rfree is the Rfactor for a subset of 5% of the reflections that were omitted from refinement. Open table in a new tab Model Building and Refinement—The structures of the mutated and/or liganded forms of PhBPL were determined by the difference Fourier synthesis based on its wild-type structure. The structures of PhBCCPΔN76 in two different crystal forms were determined by the molecular replacement with the program MOLREP (19Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4137) Google Scholar), using coordinates of the C-terminal domain of E. coli BCCP (EcBCCP, PDB ID 1BDO) as a search model (20Athappilly F.K. Hendrickson W.A. Structure. 1995; 3: 1407-1419Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The initial phases of the protein·protein complexes were obtained by the molecular replacement with MOLREP, using the crystal structures of PhBPL*, PhBPL**, and PhBCCPΔN76 as search models. After rebuilding the initial models using QUANTA (Accelrys, San Diego, CA), several rounds of the manual model revision and the structure refinement using CNS (21Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar) were performed for all the structures. The electron densities for biotinyl-5′-AMP and biocytin clearly indicate a covalent linkage between biotin and AMP and between biotin and the Lys115 of BCCP, respectively. Refinement statistics are summarized in tables (Table 1 and supplemental Tables S1 and S2). All of the models have excellent stereochemistry, as evaluated by the program PROCHECK (22Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The structural superposition analysis was performed using the program LSQKAB (23Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2327) Google Scholar) and a subsequent classification of structural changes according to the multiple superposition method (24Kunishima N. Asada Y. Sugahara M. Ishijima J. Nodake Y. Sugahara M. Miyano M. Kuramitsu S. Yokoyama S. Sugahara M. J. Mol. Biol. 2005; 352: 212-228Crossref PubMed Scopus (48) Google Scholar). The figures illustrating these structures were prepared using the program PyMOL. 3W. L. DeLano (2002) PyMOL, DeLano Scientific, San Carlos, CA. Sequence alignments were generated by ClustalX (26Jeanmougin F. Thompson J.D. Gouy M. Higgins D.G. Gibson T.J. Trends Biochem. Sci. 1998; 23: 403-405Abstract Full Text Full Text PDF PubMed Scopus (2378) Google Scholar) and displayed with ESPript (27Gouet P. Courcelle E. Stuart D.I. Metoz F. Bioinformatics. 1999; 15: 305-308Crossref PubMed Scopus (2514) Google Scholar). Structures of PhBPL—To examine the effect of mutations R48A and R48A/K111A on the structure and reactivity of PhBPL, we determined the crystal structures of PhBPL* and PhBPL** and their liganded forms PhBPL*·biotinyl-5′-AMP, PhBPL**·biotin·adenosine, and PhBPL**·biotinol-5′-AMP (supplemental Table S1). Overall, all the structures were found isomorphous to the wild-type PhBPL (9Bagautdinov B. Kuroishi C. Sugahara M. Kunishima N. J. Mol. Biol. 2005; 353: 322-333Crossref PubMed Scopus (49) Google Scholar). The crystals contain one dimer in the asymmetric unit. The mutants have essentially the same overall protomer conformation as the wild-type PhBPL: a Cα superposition between any pair of the protomers results in the root-mean-square deviation (r.m.s.d.) value of <1 Å. The liganded forms of crystals were prepared by a cocrystallization with ligands, to avoid crystal packing effects. The apo structures of PhBPL mutants show that the active site loop Gly45-Trp53 is disordered. In the liganded structures, the loop becomes ordered and closes over the bound substrates to form the main hole of the active site. This ligand-induced ordering of the active site loop is also observed in the structures of wild-type PhBPL (9Bagautdinov B. Kuroishi C. Sugahara M. Kunishima N. J. Mol. Biol. 2005; 353: 322-333Crossref PubMed Scopus (49) Google Scholar). The cocrystallization of PhBPL* with ATP and biotin provided the PhBPL*·biotinyl-5′-AMP complex where the U-shaped biotinyl-5′-AMP was found in the bifurcated main hole of BPL*, indicating the retained functionality of the single mutant (Fig. 1A, and supplemental Table S3 Fig. S1). However, the same cocrystallization condition using PhBPL** resulted in a substrate complex where the active site hole was occupied by biotin and adenosine. Because ATP dominates the adenosine nucleotide species in the crystallization solution, it is likely that the modeled adenosine is an ordered part of ATP. The nucleotide binding mode in the double mutant was compared with that in the wild-type enzyme (supplemental Table S3 and Fig. S2): PhBPL·ATP (PDB ID 1X01) and PhBPL·ADP (PDB ID 1WNL). In the wild-type enzyme, the phosphate part of nucleotides is recognized by polar interactions with the basic residues Arg48, Arg51, Lys111, and Arg233. Thus the mutations R48A and K111A would induce the disordering of the triphosphate moiety of ATP and tend to prevent it from reacting with biotin in the mutant crystals. Most likely, this reduced reactivity of bound ATP allowed the successful cocrystallization of single and double mutants with BCCP. As described in the later section of BPL·BCCP complex, PhBPL** can produce holo-BCCP, indicating the retained functionality of the double mutant. However, the lowest ability in the reaction intermediate production may hinder the formation of biotinyl-5′-AMP-liganded crystals in the double mutant. The binding mode of the reaction intermediate in the double mutant could be estimated from the PhBPL**·biotinol-5′-AMP structure, which was determined from a cocrystal with the reaction intermediate analog biotinol-5′-AMP (supplemental Table S3 and Fig. S1). The conformation of bound biotinol-5′-AMP in PhBPL** is quite similar to that of bound biotinyl-5′-AMP in PhBPL* as well as in wild-type PhBPL, suggesting that the mutations used are not essentially defective in the formation of biotinyl-5′-AMP. Structures of PhBCCPΔN76—We designed and expressed a truncated version of PhBCCP lacking N-terminal 76 residues and containing 73 C-terminal amino acids from Val77 (PhBCCPΔN76, residues 76-149), to improve its handling and crystallizability. It is well known that the C-terminal half of biotinyl domain is expressed as a stable protein, which can be biotinylated normally both in vivo and in vitro (28Cronan Jr., J.E. J. Biol. Chem. 1990; 265: 10327-10333Abstract Full Text PDF PubMed Google Scholar, 29Chapman-Smith A. Turner D.L. Cronan Jr., J.E. Morris T.W. Wallace J.C. Biochem. J. 1994; 302: 881-887Crossref PubMed Scopus (119) Google Scholar, 30Nenortas E. Beckett D. J. Biol. Chem. 1996; 271: 7559-7567Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 31Stolz J. Ludwig A. Sauer N. FEBS Letts. 1998; 440: 213-217Crossref PubMed Scopus (25) Google Scholar, 32Leon-Del-Rio A. Gravel R.A. J. Biol. Chem. 1994; 269: 22964-22968Abstract Full Text PDF PubMed Google Scholar, 33Wood H.G. Harmon F.R. Wuhr B. Hubner K. Lynen F. J. Biol. Chem. 1980; 255: 7397-7409Abstract Full Text PDF PubMed Google Scholar). Located upstream of the biotinyl domain sequence are proline/alanine-rich sequences of varying lengths, which have been proposed to act as flexible linkers (34Cronan Jr., J.E. J. Biol. Chem. 2002; 277: 22520-22527Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The crystal structure of PhBCCPΔN76 (form I; modeled residues 76-149) has been solved by the molecular replacement using BCCP from EcBCCP (PDB ID 1BDO) as a search model and refined at a resolution of 1.55 Å (supplemental Table S2). The second form of crystal structure was determined by molecular replacement using the form I structure and refined at a resolution of 1.55 Å (form II; modeled residues 80-149). In the form I crystal, there is one protomer in the crystallographic asymmetric unit, whereas the form II crystal has two protomers in the asymmetric unit. The r.m.s.d. values from a Cα superposition between the observed three crystallographically independent protomers are <1 Å, indicating an essentially identical protomer fold. The overall fold of PhBCCPΔ76 is described as a flattened β-barrel structure comprising two four-stranded β-sheets with the N- and C-terminal residues close together at one end of the structure (Fig. 1B). The biotinyl domain has an internal 2-fold symmetry. The Cα atoms in two halves of the molecule (residues 80-112 and 117-149) can be aligned with an r.m.s.d. of 0.48 Å after a rotation about the 2-fold axis, clearly indicating that this molecule was created by a duplication of two identical ancestor genes as first suggested by Toh et al. (35Toh H. Kondo H. Tanabe T. Eur. J. Biochem. 1993; 215: 687-696Crossref PubMed Scopus (55) Google Scholar) and structurally confirmed by Athappilly and Hendrickson (20Athappilly F.K. Hendrickson W.A. Structure. 1995; 3: 1407-1419Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). From a structural perspective, it is likely that the ancestor protein is a dimer of two identical hammerhead motifs β6-β7-β8-β1 and β2-β3-β4-β5. The biotinylation target Lys115 is located at the type I’ hairpin β-turn involving two residues Met114 and Lys115, which connects the N-terminal and C-terminal halves of the biotinyl domain. The sequences of C-terminal regions corresponding to the biotinyl domain are well conserved among different BCCPs (Fig. 2A). Accordingly, the crystal structure of PhBCCPΔ76 confirms the same overall folding with the reported crystal structures of the C-terminal fragment of EcBCCP (20Athappilly F.K. Hendrickson W.A. Structure. 1995; 3: 1407-1419Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 36Roberts E.L. Shu N. Howard M.J. Broadhurst R.W. Chapman-Smith A. Wallace J.C. Cronan Jr., J.E. Perham R.N. Biochemistry. 1999; 38: 5045-5053Crossref PubMed Scopus (68) Google Scholar) and the 1.3 S subunit of the Propionibacterium shermanii transcarboxylase complex (37Reddy D.V. Shenoy B.C. Carey P.R. Sonnichsen F.D. Biochemistry. 2000; 39: 2509-2516Crossref PubMed Scopus (48) Google Scholar). Furthermore, biotinyl domains have been shown to bear sequence and structural similarities to the lipoyl domains of 2-oxo-acid dehydrogenase multienzyme complexes, which undergo an analogous post-translational modification (1Knowles J.R. Annu. Rev. Biochem. 1989; 58: 195-221Crossref PubMed Scopus (355) Google Scholar, 38Brocklehurst S.M. Perham R.N. Protein Sci. 1993; 2: 626-639Crossref PubMed Scopus (95) Google Scholar, 39Dardel F. Davis A.L. Laue E.D. Perham R.N. J. Mol. Biol. 1993; 229: 1037-1048Crossref PubMed Scopus (120) Google Scholar). There are two main differences among these structures: PhBCCP and many other BCCP structures lack the β2-β3 “thumb” loop, which is observed in EcBCCP (20Athappilly F.K. Hendrickson W.A. Structure. 1995; 3: 1407-1419Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar); the two symmetric halves of all biotinyl domain structures are connected by a type I′ β-turn, whereas the lipoyl domains adopt a type I conformation for this turn. PhBCCP may be an evolutionally more primitive form as compared with EcBCCP, because PhBCCP is more symmetric in terms of the duplication on hammerhead motifs and lacks the thumb loop providing additional recognition for the biotinyl moiety. Structures of PhBPL·PhBCCP Complex—The complex structures of PhBPL*·biotin·adenosine·PhBCCPΔN76 and PhBPL**·biotin·adenosine·holo-PhBCCPΔN76 were determined by molecular replacement using the coordinates of PhBPL*, PhBPL**, and PhBCCPΔN76 structures and refined at resolutions of 2.7 and 2.0 Å, respectively (Fig. 1C and Table 1). These two forms of PhBPL·PhBCCP complex crystals are nearly isomorphous and both have a 2:2 heterotetramer in the asymmetric unit. The PhBPL·PhBCCP association involves the formation of a large intermolecular β-sheet, which is solvent-exposed on one side to house the biotinyl-5′-AMP. Notably, the bifurcated main holes of both the complexes are occupied by biotin and adenosine. As mentioned in the first section, we suppose that the modeled adenosine is an ordered part of ATP. The B-factors for the ligand atoms are comparable to those of other atoms in the crystal structure; averaged B-factors for the ligand atoms and all atoms are 43.7 Å2 and 42.1 Å2 in the single mutant complex and 24.8 Å2 and 28.4 Å2 in the double mutant complex, respectively (Table 1 and supplemental Table S3). Judging from this fact, it is likely that the bound ligands have some biological role in the reaction mechanism rather than a mutation/crystallization artifact from the binding of low affinity ligands. In both the complexes, although the binding mode of the biotin and the adenine ring is similar to that of the corresponding part of biotinyl-5′-AMP seen in the other liganded forms, the ribose ring shows distinct conformation when compared with that of biotinyl-5′-AMP (supplemental Fig. S3). This suggests that the mutations tend to prevent ATP from reacting with biotin in the complex crystals. However, because the single mutant can provide the biotinyl-5′-AMP liganded form and the biotinylated holo-PhBCCP is found in the double mutant complex, the functionality of the mutants would be retained. Therefore, the snapshots fortunately captured at points before and after the biotinylation of PhBCCP may shed light on the interaction of proteins and the target protein/lysine residue specificity of BPL. The association of two 1:1 complexes of PhBPL·PhBCCP to make the 2:2 complex is mediated by the PhBPL dimer interface analogous to one observed in the wild-type PhBPL (9Bagautdinov B. Kuroishi C. Sugahara M. Kunishima N. J. Mol. Biol. 2005; 353: 322-333Crossref PubMed Scopus (49) Google Scholar). At the PhBPL·PhBCCP interface, buried solvent-accessible surface area is ∼900 Å2 per protomer, which is comparable to the PhBPL dimer interface area of 1030 Å2 in the complex. The intermolecular interface is mainly hydrophobic (over 60% of interface atoms are non-polar) and defined by a number of hydrogen bonds (Table 2). The electrostatic potential on the surfaces of proteins shows a heterogeneous charge distribution that reflects a good charge complementarity between the interacting surfaces (Fig. 3). This indicates that electrostatic interactions are important for the complex formation in addition to a pronounced molecular surface complementarity. The structures of the apo and holo forms of PhBCCPΔN76 in free and complex states are generally similar, suggesting that binding to PhBPL and biotinylation causes few significant changes in the overall fold of the biotinyl domain, except for the β4-β5 hairpin turn. In contrast, structures of PhBPL* and PhBPL** show more extensive local conformational changes. Although the catalytic domain is relatively similar to the one in free state, the C-terminal domain shows large variations (Fig. 4A and supplemental Fig. S4). In the present two complex structures, we observed four crystallographically independent BPL·BCCP complexes. From the overall structural similarity in the orientational relationship between BPL and BCCP, the four independent complexes can be classified into three groups (Fig. 4A and supplemental Table S4). The B and D subunits of the complexes are in a nearly identical spatial relationship, in which the substrate lysine of BCCP enters close to the BPL active site. We designate this state as the formation stage 1. The A and C subunits of both the complexes are also in closely related orientations. In the single mutant complex, BCCP seems to approach the BPL active site in another way; this state is referred to as the formation stage 2. The designation of “stage 1” or “stage 2” does not imply an order in which these putative snapshots of the reaction occur along the reaction coordinate. On the other hand, in the double mutant complex, the biotinylated holo-BCCP is observed as the reaction product; this state is referred to as the product stage. Thus, there is a certain amount of plasticity around the active site of PhBPL to accommodate its substrate PhBCCP. Especially, a conformational flexibility of a C-terminal domain loop (Ile226-Asp229) in the side hole to the active site provides an entry for the substrate lysine residue and an exit pathway for the product biocytin residue of holo-BCCP (Fig. 1C). Notably, the loop residue Tyr227 undergoes an open/close motion, which may act as a lid of the side hole to regulate the traffic of ligands: open in the formation stage 1 and closed in the other stages (Fig. 4B). In addition, the induced-fit ordering of the active site loop (Gly45-Trp53) upon binding substrat" @default.
• W2010392743 created "2016-06-24" @default.
• W2010392743 creator A5013415378 @default.
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• W2010392743 title "Protein Biotinylation Visualized by a Complex Structure of Biotin Protein Ligase with a Substrate" @default.
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