Binding-induced Conformational Changes Research Articles

A Key Hydrophobic Patch Identified in an AAA+ Protein Essential for Its In Trans Inhibitory Regulation

Bacterial enhancer binding proteins (bEBPs) are a subclass of the AAA+ (ATPases Associated with various cellular Activities) protein family. They are responsible for σ54-dependent transcription activation during infection and function under many stressful growth conditions. The majority of bEBPs are regulated in their formation of ring-shaped hexameric self-assemblies via an amino-terminal domain through its phosphorylation or ligand binding. In contrast, the Escherichia coli phage shock protein F (PspF) is negatively regulated in trans by phage shock protein A (PspA). Up to six PspA subunits suppress PspF hexamer action. Here, we present biochemical evidence that PspA engages across the side of a PspF hexameric ring. We identify three key binding determinants located in a surface-exposed ‘W56 loop’ of PspF, which form a tightly packed hydrophobic cluster, the ‘YLW’ patch. We demonstrate the profound impact of the PspF W56 loop residues on ATP hydrolysis, the σ54 binding loop 1, and the self-association interface. We infer from single-chain studies that for complete PspF inhibition to occur, more than three PspA subunits need to bind a PspF hexamer with at least two binding to adjacent PspF subunits. By structural modelling, we propose that PspA binds to PspF via its first two helical domains. After PspF binding-induced conformational changes, PspA may then share structural similarities with a bEBP regulatory domain.

Label-Free and Substrate-Free Potentiometric Aptasensing Using Polycation-Sensitive Membrane Electrodes

A potentiometric label-free and substrate-free (LFSF) aptasensing strategy which eliminates the labeling, separation, and immobilization steps is described in this paper. An aptamer binds specifically to a target molecule via reaction incubation, which could induce a change in the aptamer conformation from a random coil-like configuration to a rigid folded structure. Such a target binding-induced aptamer conformational change effectively prevents the aptamer from electrostatically interacting with the protamine binding domain. This could either shift the response curve for the potentiometric titration of the aptamer with protamine as monitored by a conventional polycation-sensitive membrane electrode or change the current-dependent potential detected by a protamine-conditioned polycation-sensitive electrode with the pulsed current-driven ion fluxes of protamine across the polymeric membrane. Using adenosine triphosphate (ATP) as a model analyte, the proposed concept offers potentiometric detection of ATP down to the submicromolar concentration range and has been applied to the determination of ATP in HeLa cells. In contrast to the current LFSF aptasensors based on optical detection, the proposed strategy allows the LFSF biosensing of aptamer/target binding events in a homogeneous solution via electrochemical transduction. It is anticipated that the proposed strategy will lay a foundation for development of potentiometric sensors for LFSF aptasensing of a variety of analytes where target binding-induced conformational changes such as the formation of folded structures and the opening of DNA hairpin loops are involved.

Aptasensor based on triplex switch for SERS detection of cytochrome c

We report a sensitive approach for SERS detection of cytochrome c using target binding-induced conformational changes of signal transduction probe (STP). STP labeled with a SERS-active molecule, carboxy-X-rhodamine (ROX), is immobilized on the substrate where the formation of a rigid triplex switching structure with aptamers does not allow SERS amplification to take place. The target binding event leads to an enhancement in SERS intensity of ROX adsorbed on the gold surface. Meanwhile, we found that an appropriate STP surface density could shield the SERS signal produced by protein adsorption which would foul the sensing surface. In addition, cytochrome c aptamers used were not the original sequence but reorganized in the nonspecific binding site to adapt to our design. This method provides a low detection limit of 2 nM (10 fmol within 5 μL sample solution), and shows good selectivity toward cytochrome c compared to interfering proteins such as hemoglobin and immunoglobulin G. The general strategy of the method can also be extended to aptamer or DNA based sensors.

MBNL1-RNA Interactions: Binding-Induced Rna Conformational Changes

The muscleblind-like protein 1 (MBNL1) is an RNA binding protein that regulates mRNA alternative splicing. It is implicated to be involved in myotonic dystrophy, an inherited multisystem disease, through interacting with poly(CUG) or poly(CCUG) RNA. In the disease state, MBNL1 is abnormally expressed and translocated, which leads to many mis-splicing events. MBNL1 can bind various RNA substrates, most of which share a stem-loop structure containing 5’YGCY3’(Y=U or C) motifs. Using FRET together with a combination of stopped-flow kinetic fluorescence measurements and gel shift assays, we have investigated the relationship between MBNL1-RNA interactions and RNA secondary structure. This work suggested that binding of MBNL1 destabilizes the RNA secondary structure and leads to a partial melting of the stem loop RNA. The RNA secondary structure stability is directly correlated with the kinetic and thermodynamic properties of the MBNL1-RNA complex. The data presented here provides insight into the role of RNA secondary structure in regulation of alternative splicing by MBNL1 and suggests new perspectives for drug development for myotonic dystrophy.

On the use of distance constraints in protein-protein docking computations

Protein-protein docking (PPD) is a computational process that predicts the structure of a complex of two interacting proteins from their unbound structures. The accuracy of PPD predictions is low, but can be greatly enhanced if experimentally determined distance data are available for incorporation into the prediction. However, the specific effects of distance constraints on PPD predictions are largely uncharacterized. In this study, we systematically simulated the effects of using distance constraints both on a new distance constraint-driven PPD approach, called DPPD, and also, by re-ranking, on a well-established grid-based global search approach. Our results for a PPD benchmark dataset of 84 protein complexes of known structures showed that near 100% docking success rates could be obtained when the number of distance constraints exceeded six, the degrees of freedom of the system, but the success rate was significantly reduced by long distance constraints, large binding-induced conformational changes, and large errors in the distance data. Our results also showed that, under most conditions simulated, even two or three distance constraints were sufficient to achieve a much better success rate than those using a sophisticated physicochemical function to re-rank the results of the global search. Our study provides guidelines for the practical incorporation of experimental distance data to aid PPD predictions.

Relative Solvent Accessible Surface Area Predicts Protein Conformational Changes upon Binding

SummaryProtein interactions are often accompanied by significant changes in conformation. We have analyzed the relationships between protein structures and the conformational changes they undergo upon binding. Based upon this, we introduce a simple measure, the relative solvent accessible surface area, which can be used to predict the magnitude of binding-induced conformational changes from the structures of either monomeric proteins or bound subunits. Applying this to a large set of protein complexes suggests that large conformational changes upon binding are common. In addition, we observe considerable enrichment of intrinsically disordered sequences in proteins predicted to undergo large conformational changes. Finally, we demonstrate that the relative solvent accessible surface area of monomeric proteins can be used as a simple proxy for protein flexibility. This reveals a powerful connection between the flexibility of unbound proteins and their binding-induced conformational changes, consistent with the conformational selection model of molecular recognition.

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry for Probing the Microenvironment of Histidine Residues in Dihydrofolate Reductase

BackgroundHistidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C2-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.Methodology/Principal FindingsUsing His-HDX-MS, the pK a values and the half-lives (t 1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK a and t 1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK a, t 1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK a and t 1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.Conclusions/SignificanceOur results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.

Switch-based biosensors: a new approach towards real-time, in vivo molecular detection.

Although the ability to monitor specific molecules in vivo in real-time could revolutionize many aspects of healthcare, the technological challenges that stand in the way of reaching this goal are considerable and are poorly met by most existing analytical approaches. Nature, however, has already solved the problem of real-time molecular detection in complex media by employing biomolecular "switches". That is, protein and nucleic acids that sense chemical cues and, by undergoing specific, binding-induced conformational changes, transduce this recognition into high-gain signal outputs. Here, we argue that devices that employ such switches represent a promising route towards versatile, real-time molecular monitoring in vivo.

Abstract 715: Synthesis of potent anticancer genistein derivatives to cure estrogen receptor-related breast cancers

Abstract Abstract: Background: Estrogen Receptor (ER) is a major target for the treatment of breast cancer as estrogens act to stimulate the growth of estrogen-dependent tumors. Estrogens exert their physiological action through ER (ERα and ERβ). Both of them are expressed in normal breast tissue and several other tissues. In many breast cancers, an up-regulation of ERα expression as well as down-regulation of ERβ expression was observed by several groups. Studies also showed that high ERβ levels are associated with a reduced breast cancer risk. Genistein is a soy isoflavone with a structure similar to estrogen, can mimic and antagonize the estrogen effects in tissues, which could allow it to act as chemo-preventive agent in breast cancer. It has been noted that Genistein is an effective anticancer agent in breast cancer cell lines, but only at supraphysiological concentrations. We synthesized several structurally modified Genistein derivatives, to enhance its antiproliferative effects and to minimize its alpha-estrogenic effects. Methods: We first synthesized 7 Genistein derivatives (MA-6, MA-8, MA-11, MA-19, MA-20, MA-21 and MA-22) in which Genistein was linked to aliphatic side-chains or heterocyclic triazole moieties that precluded standard agonist binding-induced conformational changes in the ERα receptor pocket. Then we investigated the biochemical effects of these substituted genistein derivatives in the three ER positive breast cancer cell lines (MCF7, 21PT and T47D). Effects were compared to those seen with the parent compound genistein. Results: 1) Three derivatives (MA-6, MA-8 and MA-19) showed growth inhibitory activity at 5-15 times lower concentrations than the parent Genistein, as assessed by 50% inhibitory concentrations (IC50) in all the 3 breast cancer cell lines. They also induced apoptosis in the breast cancer cell lines as it was reported earlier by Genistein. 2) Treatment with these 3 genistein derivatives in the above low IC50 concentrations, resulted in a decrease of ERα levels and an increase in ERβ levels, at similar levels compared to Genistein itself. These effects were accompanied by the expected changes in gene expression of presenilin-2 and psoriasin, downstream genes of ERα and ERβ signaling respectively. 3) The observed increase of ER β /ER α ratio after MA8, MA6 and MA19 treatments also reached to similar levels achieved by Genistein, explain their high and better potency as anticancer drugs. Conclusion: Modified Genistein derivatives MA6, MA8 and MA19 reduce proliferation & ERα levels and induce ERβ levels in all the 3 estrogen positive breast cancer cell lines. These effects are seen at 5-15 times lower IC50 concentrations than the parent drug Genistein. ERβ level enhancement by the genistein derivatives may also be associated with a reduced breast cancer risk by the modulation of growth. These new drugs may therefore be much better chemopreventive agents than Genistein itself. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 715.

Probing the Role of Backbone Hydrogen Bonding in a Critical β Sheet of the Extracellular Domain of a Cys‐Loop Receptor

Probing the sheet: The network of hydrogen bonds formed in the outer beta sheet of the nicotinic acetylcholine receptor (nAChR; see figure) is fairly robust and tolerates single amide-to-ester mutations throughout. However, eliminating two proximal hydrogen bonds completely destroys receptor function; this adds further support to gating models that ascribe important roles to these beta strands of the nAChR extracellular domain.Long-range communication is essential for the function of members of the Cys-loop family of neurotransmitter-gated ion channels. The involvement of the peptide backbone in binding-induced conformational changes that lead to channel gating in these membrane proteins is an interesting, but unresolved issue. To probe the role of the peptide backbone, we incorporated a series of alpha-hydroxy acid analogues into the beta-sheet-rich extracellular domain of the muscle subtype of the nicotinic acetylcholine receptor, the prototypical Cys-loop receptor. Specifically, mutations were made in beta strands 7 and 10 of the alpha subunit. A number of single backbone mutations in this region were well tolerated. However, simultaneous introduction of two proximal backbone mutations led to surface-expressed, nonfunctional receptors. Together, these data suggest that while the receptor is remarkably robust in its ability to tolerate single amide-to-ester mutations throughout these beta strands, more substantial perturbations to this region have a profound effect on the protein. These results support a model in which backbone movements in the outer beta sheet are important for receptor function.

Conformational changes within the HLA‐A1:MAGE‐A1 complex induced by binding of a recombinant antibody fragment with TCR‐like specificity

Although there is X-ray crystallographic evidence that the interaction between major histocompatibility complex (MHC, in humans HLA) class I molecules and T cell receptors (TCR) or killer cell Ig-like receptors (KIR) may be accompanied by considerable changes in the conformation of selected residues or even entire loops within TCR or KIR, conformational changes between receptor-bound and -unbound MHC class I molecules of comparable magnitude have not been observed so far. We have previously determined the structure of the MHC class I molecule HLA-A1 bound to a melanoma antigen-encoding gene (MAGE)-A1-derived peptide in complex with a recombinant antibody fragment with TCR-like specificity, Fab-Hyb3. Here, we compare the X-ray structure of HLA-A1:MAGE-A1 with that complexed with Fab-Hyb3 to gain insight into structural changes of the MHC molecule that might be induced by the interaction with the antibody fragment. Apart from the expulsion of several water molecules from the interface, Fab-Hyb3 binding results in major rearrangements (up to 5.5 A) of heavy chain residues Arg65, Gln72, Arg145, and Lys146. Residue 65 is frequently and residues 72 and 146 are occasionally involved in TCR binding-induced conformational changes, as revealed by a comparison with MHC class I structures in TCR-liganded and -unliganded forms. On the other hand, residue 145 is subject to a reorientation following engagement of HLA-Cw4 and KIR2DL1. Therefore, conformational changes within the HLA-A1:MAGE-A1:Fab-Hyb3 complex include MHC residues that are also involved in reorientations in complexes with natural ligands, pointing to their central importance for the peptide-dependent recognition of MHC molecules.

Structural Analysis of the Interactions Between Paxillin LD Motifs and α-Parvin

SummaryThe adaptor protein paxillin contains five conserved leucine-rich (LD) motifs that interact with a variety of focal adhesion proteins, such as α-parvin. Here, we report the first crystal structure of the C-terminal calponin homology domain (CHC) of α-parvin at 1.05 Å resolution and show that it is able to bind all the LD motifs, with some selectivity for LD1, LD2, and LD4. Cocrystal structures with these LD motifs reveal the molecular details of their interactions with a common binding site on α-parvin-CHC, which is located at the rim of the canonical fold and includes part of the inter-CH domain linker. Surprisingly, this binding site can accommodate LD motifs in two antiparallel orientations. Taken together, these results reveal an unusual degree of binding degeneracy in the paxillin/α-parvin system that may facilitate the assembly of dynamic signaling complexes in the cell.

Flexibility and dynamics of NhaA Na +/H +-antiporter of Escherichia coli studied by Fourier transform infrared spectroscopy

NhaA (41,355 Da) is a Na +/H + antiporter of Escherichia coli which plays a central role in regulation of intracellular pH, cellular Na + content, and cell volume [E. Padan, S. Schuldiner, J. Exp. Biol. 196 (1994) 443]. Its activity is strongly regulated by pH and increases over 3 orders of magnitude between pH 7 and 8 [A. Rothman, Y. Gerchman, E. Padan, S. Schuldiner, Biochemistry 36 (1997) 14572]. Protein dynamics and flexibility in the activated and inactivated state, respectively, was analysed by probing accessibility in 1H/ 2H exchange experiments for the wild type and the mutant G338S which is constitutively active independent of pH [A. Rimon, Y. Gerchman, Z. Kariv, E. Padan, J. Biol. Chem. 273 (1998) 26470]. This was studied by ATR–FTIR difference spectroscopy using a home built microvolume (<5 μl) perfusion chamber [E. Agić, O. Klein, W. Mäntele, Proceedings of the 10th European Conference on the Spectroscopy of Biological Molecules, vol. 93, 2003, ISBN 9634826148; S. Gourion-Arsiquaud, S. Chevance, P. Bouyer, L. Garnier, J.-L. Montillet, A. Bondon, C. Berthomieu, Biochemistry 44 (2005) 8652]. The solution or suspension of the target protein is contained in a chamber with sample volumes of below 5 μl. It is in contact with the ATR crystal and separated from the flowing effector molecules by a dialysis membrane. The flow-ATR unit is characterised by high stability, fast response, and high sensitivity for the IR spectroscopic detection of binding-induced conformational changes and reactions. On the basis of 1H– 2H exchange of NhaA followed in the amide I and amide II region of the IR spectrum, it is concluded that the accessible fraction of the polypeptide chain of NhaA increases by more than 10% in the active state. For the mutant, no changes in accessibility were observed for different pH values. The increase of Na + concentration increases the extent of exchange. The stability of the wild type protein in the active and inactive form was analysed by measuring the temperature profiles of the IR spectra. A decrease of the structural stability of the protein with activation was observed. Together with the results from 1H/ 2H exchange, the inactive state represents a more compact form whereas activation induces a more open conformation of the protein.

Ultrasensitive label-free aptamer-based electronic detection.

Ultrasensitive label-free aptamer-based electronic detection.

Voltage-independent Sodium-binding Events Reported by the 4B-4C Loop in the Human Glutamate Transporter Excitatory Amino Acid Transporter 3

Glutamate transporters (excitatory amino acid transporter (EAATs)) are critical for normal excitatory signaling and maintaining subtoxic glutamate concentrations in mammalian central nervous system. Recently, a crystal structure for a homologous transporter in bacteria was reported. Still, little is understood regarding the mechanism of substrate uptake. In transmembrane domain 4, the mammalian EAATs contain a stretch of over 50 amino acids (4B-4C loop) that are absent in the bacterial protein. These residues have been suggested to be located in the large extracellular vestibule seen in the crystal structure. State-dependent trypsin-cleavage sites have been reported in this region, suggesting that the 4B-4C loop undergoes significant conformational changes. Here we employed substituted cysteine accessibility, voltage clamp fluorometry, and fluorescence resonance energy transfer on oocytes expressing mutant EAAT3 transporters to determine the location and functionality of the 4B-4C loop. We find that this loop extends from near the center of the protein and that the majority of the residues are positioned on the outer perimeter of the protein, rather than inside the vestibule. Our fluorescence resonance energy transfer measurements demonstrated that these residues do not undergo large scale motions during glutamate uptake. However, our voltage clamp fluorometry studies indicate that these residues report on Na(+) and glutamate binding-induced conformational changes, including a previously un-described voltage-independent component of Na(+) binding to the transporter. The finding that residues far from the glutamate-binding site report on several different types of binding events suggests that the series of small conformational changes that accomplish glutamate uptake extend throughout the transporter structure.

Structural Basis for p53 Binding-induced DNA Bending

Specific p53 binding-induced DNA bending has important biological implications such as transcription activation. However, the detailed structures of the bent DNA and the p53-DNA complex are still unavailable, hampering our understanding of the mechanism for p53-induced DNA bending and its consequent biological significance. To gain insight into the p53 binding-induced DNA bending, we performed molecular dynamics simulations on DNA segments with the consensus sequence for p53-specific binding, half site DNA-p53 complexes, and full site DNA-p53 complexes. We show that each DNA-bound p53 core domain caused a local DNA conformational change within the quarter site; upon the binding of the p53 dimer, there was an apparent DNA bending at the center of the half site; when bound with two p53 dimers, the full site DNAs with two different sequences bent 20 and 35 degrees, respectively. These results are in agreement with experimental observations. Our simulations demonstrate that the two p53 dimers favored a staggered conformation in which they make favorable interactions at the interface. This dimer-dimer interface organization necessitated conformational changes in the DNA, leading to the bending at the center of the full site, which in turn is dependent on the DNA sequence. Overall, our results provide the detailed atomic model for the DNA-p53 tetramer complex and delineate the roles of DNA-p53, p53 dimer-dimer interactions, and DNA sequence in specific p53 binding-induced DNA conformational changes.

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.

Comparison of Ligand-induced Conformational Changes and Domain Closure Mechanisms, Between Prokaryotic and Eukaryotic Dehydroquinate Synthases

Dehydroquinate synthase (DHQS) is a potential target for the development of novel broad-spectrum antimicrobial drugs, active against both prokaryotes and lower eukaryotes. Structures have been reported for Aspergillus nidulans DHQS (AnDHQS) in complexes with a range of ligands. Analysis of these AnDHQS structures showed that a large-scale domain movement occurs during the normal catalytic cycle, with a complex series of structural elements propagating substrate binding-induced conformational changes away from the active site to distal locations. Compared to corresponding fungal enzymes, DHQS from bacterial species are both mono-functional and significantly smaller. We have therefore determined the structure of Staphylococcus aureus DHQS (SaDHQS) in five liganded states, allowing comparison of ligand-induced conformational changes and mechanisms of domain closure between fungal and bacterial enzymes. This comparative analysis shows that substrate binding initiates a large-scale domain closure in both species' DHQS and that the active site stereochemistry, of the catalytically competent closed-form enzyme thus produced, is also highly conserved. However, comparison of AnDHQS and SaDHQS open-form structures, and analysis of the putative dynamic processes by which the transition to the closed-form states are made, shows a far lower degree of similarity, indicating a significant structural divergence. As a result, both the nature of the propagation of conformational change and the mechanical systems involved in this propagation are quite different between the DHQSs from the two species.

Conversion of the allosteric transition of GroEL from concerted to sequential by the single mutation Asp-155 -> Ala.

The reaction cycle of the double-ring chaperonin GroEL is driven by ATP binding that takes place with positive cooperativity within each seven-membered ring and negative cooperativity between rings. The positive cooperativity within rings is due to ATP binding-induced conformational changes that are fully concerted. Herein, it is shown that the mutation Asp-155 --> Ala leads to an ATP-induced break in intra-ring and inter-ring symmetry. Electron microscopy analysis of single-ring GroEL particles containing the Asp-155 --> Ala mutation shows that the break in intra-ring symmetry is due to stabilization of allosteric intermediates such as one in which three subunits have switched their conformation while the other four have not. Our results show that eliminating an intra-subunit interaction between Asp-155 and Arg-395 results in conversion of the allosteric switch of GroEL from concerted to sequential, thus demonstrating that its allosteric behavior arises from coupled tertiary conformational changes.

Ligand binding-induced conformational changes in riboflavin kinase: structural basis for the ordered mechanism.

Riboflavin kinase (RFK) is an essential enzyme catalyzing the phosphorylation of riboflavin (vitamin B(2)) in the presence of ATP and Mg(2+) to form the active cofactor FMN, which can be further converted to FAD. Previously, the crystal structures of RFKs from human and Schizosaccharomyces pombe have been determined in the apo form and in complex with MgADP. These structures revealed that RFK adopts a novel kinase fold and utilizes a unique nucleotide binding site. The structures of the flavin-bound RFK obtained by soaking pre-existing crystals were also reported. Because of crystal packing restraints, these flavin-bound RFK complexes adopt conformations nearly identical with that of corresponding flavin-free structures. Here we report the structure of human RFK cocrystallized with both MgADP and FMN. Drastic conformational changes associated with flavin binding are observed primarily at the so-called Flap I and Flap II loop regions. As a result, the bound FMN molecule now interacts with the enzyme extensively and is well-ordered. Residues from Flap II interact with Flap I and shield the FMN molecule from the solvent. The conformational changes in Flap I resulted in a new Mg(2+) coordination pattern in which a FMN phosphate oxygen and Asn36 side chain carbonyl are directly coordinating to the Mg(2+) ion. The proposed catalytic base Glu86 is well-positioned for activation of the O5' hydroxyl group of riboflavin for the phosphoryl transfer reaction. The structural data obtained so far on human and yeast RFK complexes provide a rationale for the ordered kinetic mechanism of RFK.