Structure and Function of Gli123 Involved in Mycoplasma mobile Gliding.

Mycoplasma mobile is a fish pathogen that glides on solid surfaces by means of its own gliding machinery composed of internal and surface structures. In the present study, we focused on the function and structure of Gli123, a surface protein that is essential for the localization of other surface proteins. The amino acid sequence of Gli123, which is 1,128 amino acids long, contains lipoprotein-specific repeats. We isolated the native Gli123 protein from M. mobile cells and a recombinant protein, rGli123, from Escherichia coli. The isolated rGli123 complemented a nonbinding and nongliding mutant of M. mobile that lacked Gli123. Circular dichroism and rotary-shadowing electron microscopy (EM) showed that rGli123 has a structure that is not significantly different from that of the native protein. Rotary-shadowing EM suggested that Gli123 adopts two distinct globular and rod-like structures, depending on the ionic strength of the solution. Negative-staining EM coupled with single-particle analysis revealed that Gli123 forms a globular structure featuring a small protrusion with dimensions of approximately 15.7, 14.7, and 14.1 nm for the "height," major axis and minor axis, respectively. Small-angle X-ray scattering analyses indicated a rod-like structure composed of several tandem globular domains with total dimensions of approximately 34 nm in length and 6 nm in width. Both molecular structures were suggested to be dimers, based on the predicted molecular size and structure. Gli123 may have evolved by multiplication of repeating lipoprotein units and acquired a role for Gli521 and Gli349 assembly. IMPORTANCE Mycoplasmas are pathogenic bacteria that are widespread in animals. They are characterized by small cell and genome sizes but are equipped with unique abilities for infection, such as surface variation and gliding. Here, we focused on a surface-localizing protein named Gli123 that is essential for Mycoplasma mobile gliding. This study suggested that Gli123 undergoes drastic conformational changes between its rod-like and globular structures. These changes may be caused by a repetitive structure common in the surface proteins that is responsible for the modulation of the cell surface structure and related to the assembly process for the surface gliding machinery. An evolutionary process for surface proteins essential for this mycoplasma gliding was also suggested in the present study.

Recent advances in the improvement of cyanobacterial enzymes for bioalkane production.

The use of biologically produced alkanes has attracted considerable attention as an alternative energy source to petroleum. In 2010, the alkane synthesis pathway in cyanobacteria was found to include two small globular proteins, acyl-(acyl carrier protein [ACP]) reductase (AAR) and aldehyde deformylating oxygenase (ADO). AAR produces fatty aldehydes from acyl-ACPs/CoAs, which are then converted by ADO to alkanes/alkenes equivalent to diesel oil. This discovery has paved the way for alkane production by genetically modified organisms. Since then, many studies have investigated the reactions catalyzed by AAR and ADO. In this review, we first summarize recent findings on structures and catalytic mechanisms of AAR and ADO. We then outline the mechanism by which AAR and ADO form a complex and efficiently transfer the insoluble aldehyde produced by AAR to ADO. Furthermore, we describe recent advances in protein engineering studies on AAR and ADO to improve the efficiency of alkane production in genetically engineered microorganisms such as Escherichia coli and cyanobacteria. Finally, the role of alkanes in cyanobacteria and future perspectives for bioalkane production using AAR and ADO are discussed. This review provides strategies for improving the production of bioalkanes using AAR and ADO in cyanobacteria for enabling the production of carbon-neutral fuels.

The retrograde signaling protein GUN1 regulates tetrapyrrole biosynthesis.

The biogenesis of the photosynthetic apparatus in developing seedlings requires the assembly of proteins encoded on both nuclear and chloroplast genomes. To coordinate this process there needs to be communication between these organelles, but the retrograde signals by which the chloroplast communicates with the nucleus at this time are still essentially unknown. The Arabidopsis thaliana genomes uncoupled (gun) mutants, that show elevated nuclear gene expression after chloroplast damage, have formed the basis of our understanding of retrograde signaling. Of the 6 reported gun mutations, 5 are in tetrapyrrole biosynthesis proteins and this has led to the development of a model for chloroplast-to-nucleus retrograde signaling in which ferrochelatase 1 (FC1)-dependent heme synthesis generates a positive signal promoting expression of photosynthesis-related genes. However, the molecular consequences of the strongest of the gun mutants, gun1, are poorly understood, preventing the development of a unifying hypothesis for chloroplast-to-nucleus signaling. Here, we show that GUN1 directly binds to heme and other porphyrins, reduces flux through the tetrapyrrole biosynthesis pathway to limit heme and protochlorophyllide synthesis, and can increase the chelatase activity of FC1. These results raise the possibility that the signaling role of GUN1 may be manifested through changes in tetrapyrrole metabolism, supporting a role for tetrapyrroles as mediators of a single biogenic chloroplast-to-nucleus retrograde signaling pathway.

The Wako-Saitô-Muñoz-Eaton Model for Predicting Protein Folding and Dynamics.

Despite the recent advances in the prediction of protein structures by deep neutral networks, the elucidation of protein-folding mechanisms remains challenging. A promising theory for describing protein folding is a coarse-grained statistical mechanical model called the Wako-Saitô-Muñoz-Eaton (WSME) model. The model can calculate the free-energy landscapes of proteins based on a three-dimensional structure with low computational complexity, thereby providing a comprehensive understanding of the folding pathways and the structure and stability of the intermediates and transition states involved in the folding reaction. In this review, we summarize previous and recent studies on protein folding and dynamics performed using the WSME model and discuss future challenges and prospects. The WSME model successfully predicted the folding mechanisms of small single-domain proteins and the effects of amino-acid substitutions on protein stability and folding in a manner that was consistent with experimental results. Furthermore, extended versions of the WSME model were applied to predict the folding mechanisms of multi-domain proteins and the conformational changes associated with protein function. Thus, the WSME model may contribute significantly to solving the protein-folding problem and is expected to be useful for predicting protein folding, stability, and dynamics in basic research and in industrial and medical applications.

Repressor Activity of SqrR, a Master Regulator of Persulfide-Responsive Genes, Is Regulated by Heme Coordination.

Reactive sulfur species (RSS) are involved in bioactive regulation via persulfidation of proteins. However, how cells regulate RSS-based signaling and RSS metabolism is poorly understood, despite the importance of universal regulation systems in biology. We previously showed that the persulfide-responsive transcriptional factor SqrR acts as a master regulator of sulfide-dependent photosynthesis in proteobacteria. Here, we demonstrated that SqrR also binds heme at a near one-to-one ratio with a binding constant similar to other heme-binding proteins. Heme does not change the DNA-binding pattern of SqrR to the target gene promoter region; however, DNA-binding affinity of SqrR is reduced by the binding of heme, altering its regulatory activity. Circular dichroism spectroscopy clearly showed secondary structural changes in SqrR by the heme binding. Incremental change in the intracellular heme concentration is associated with small, but significant reduction in the transcriptional repression by SqrR. Overall, these results indicate that SqrR has an ability to bind heme to modulate its DNA-binding activity, which may be important for the precise regulation of RSS metabolism in vivo.

Electrostatic interactions at the interface of two enzymes are essential for two-step alkane biosynthesis in cyanobacteria.

Cyanobacterial alkane biosynthesis is catalyzed by acyl-(acyl carrier protein (ACP)) reductase (AAR) and aldehyde-deformylating oxygenase (ADO) in a two-step reaction. AAR reduces acyl-ACPs to fatty aldehydes, which are then converted by ADO to alkanes, the main components of diesel fuel. Interaction between AAR and ADO allows AAR to efficiently deliver the aldehyde to ADO. However, this interaction is poorly understood. Here, using analytical size-exclusion chromatography (SEC), we show that electrostatic interactions play an important role in the binding of the two enzymes. Alanine-scanning mutagenesis at charged residues around the substrate entry site of ADO revealed that E201A mutation greatly reduced hydrocarbon production. SEC measurement of the mutant demonstrated that E201 of ADO is essential for the AAR-ADO interaction. Our results suggest that AAR binds to the substrate entrance gate of ADO and thereby facilitates the insertion of the reactive and relatively insoluble aldehyde into the hydrophobic channel of ADO.Abbreviations: AAR: acyl-ACP reductase; ACP: acyl carrier protein; ADO: aldehyde-deformylating oxygenase; ASA: solvent accessible surface area; BSA: bovine serum albumin; CD: circular dichroism; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; GC-MS: gas chromatography-mass spectrometer; HPLC: high-performance liquid chromatography; IPTG: isopropyl-ß-D-thiogalactoside; MRE: mean residue ellipticity; NpAAR: AAR from Nostoc punctiforme PCC 73102; NpADO: ADO from Nostoc punctiforme PCC 73102; PmADO: ADO from Prochlorococcus marinus MIT 9313; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SeAAR: AAR from Synechococcus elongatus PCC 7942; SeADO: ADO from Synechococcus elongatus PCC 7942; SEC: size-exclusion chromatography; TeAAR: AAR from Thermosynechococcus elongatus BP-1; TeADO: ADO from Thermosynechococcus elongatus BP-1; UV: ultraviolet.

Conformational diversity in the intrinsically disordered HIV-1 Tat protein induced by zinc and pH.

Human immunodeficiency virus type-1 (HIV-1) transactivator of transcription (Tat) is an intrinsically disordered protein that exerts multiple functions, including activation of HIV-1 replication and induction of T-cell apoptosis and cytokine secretion via zinc binding and cellular uptake by endocytosis. However, the effects of zinc and endosomal low pH on the structure of isolated Tat protein are poorly understood. Here, we purified a monomeric zinc-bound Tat and studied its structure and acid denaturation by circular dichroism, NMR, and small-angle X-ray scattering. We found that at pH 7, the zinc-bound Tat was in a pre-molten globule state; it exhibited largely disordered conformations with residual helices and was slightly more compact than the fully unfolded states that were observed at pH 4 or in the zinc-free form. Moreover, acid-induced unfolding transitions in secondary structure and molecular size occurred at different pH ranges, indicating the presence of an expanded and helical intermediate at pH ∼6. Taken together, the extent of structural disorder in the intrinsically disordered Tat protein is highly sensitive to zinc and pH, suggesting that zinc binding and pH affect Tat structures and thereby control the versatile functions of Tat.

Production and characterization of recombinant P1 adhesin essential for adhesion, gliding, and antigenic variation in the human pathogenic bacterium, Mycoplasma pneumoniae.

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6 × His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.

Mutational analysis of a catalytically important loop containing active site and substrate-binding site in Escherichia coli phytase AppA.

A phytase from Escherichia coli, AppA, has been the target of protein engineering to reduce the amount of undigested phosphates from livestock manure by making phosphorous from phytic acid available as a nutrient. To understand the contribution of each amino acid in the active site loop to the AppA activity, alanine and glycine scanning mutagenesis was undertaken. The results of phytase activity assay demonstrated loss of activity by mutations at charged residues within the conserved motif, supporting their importance in catalytic activity. In contrast, both conserved, non-polar residues and non-conserved residues tended to be tolerant to Ala and/or Gly mutations. Correlation analyses of chemical/structural characteristics of each mutation site against mutant activity revealed that the loop residues located closer to the substrate have greater contribution to the activity of AppA. These results may be useful in efficiently engineering AppA to improve its catalytic activity. Abbreviations: AppA: pH 2.5 acid phosphatase; CSU: contacts of structural units; HAPs: histidine acid phosphatases; SASA: solvent accessible surface area; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SSM: site-saturation mutagenesis; WT: wild type.

Cyanobacterial Enzymes for Bioalkane Production.

Cyanobacterial biosynthesis of alkanes is an attractive way of producing substitutes for petroleum-based fuels. Key enzymes for bioalkane production in cyanobacteria are acyl-ACP reductase (AAR) and aldehyde-deformylating oxygenase (ADO). AAR catalyzes the reduction of the fatty acyl-ACP/CoA substrates to fatty aldehydes, which are then converted into alkanes/alkenes by ADO. These enzymes have been widely used for biofuel production by metabolic engineering of cyanobacteria and other organisms. However, both proteins, particularly ADO, have low enzymatic activities, and their catalytic activities are desired to be improved for use in biofuel production. Recently, progress has been made in the basic sciences and in the application of AAR and ADO in alkane production. This chapter provides an overview of recent advances in the study of the structure and function of AAR and ADO, protein engineering of these enzymes for improving activity and modifying substrate specificities, and examples of metabolic engineering of cyanobacteria and other organisms using AAR and ADO for biofuel production.

Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding.

Intrinsically disordered proteins (IDPs) frequently function in protein interaction networks that regulate crucial cellular signaling pathways. Many IDPs undergo transitions from disordered conformational ensembles to folded structures upon binding to their cellular targets. Several possible binding mechanisms for coupled folding and binding have been identified: folding of the IDP after association with the target ("induced fit"), or binding of a prefolded state in the conformational ensemble of the IDP to the target protein ("conformational selection"), or some combination of these two extremes. The interaction of the intrinsically disordered phosphorylated kinase-inducible domain (pKID) of the cAMP-response element binding (CREB) protein with the KIX domain of a general transcriptional coactivator CREB-binding protein (CBP) provides an example of the induced-fit mechanism. Here we show by NMR relaxation dispersion experiments that a different intrinsically disordered ligand, the transactivation domain of the transcription factor c-Myb, interacts with KIX at the same site as pKID but via a different binding mechanism that involves elements of conformational selection and induced fit. In contrast to pKID, the c-Myb activation domain has a strong propensity for spontaneous helix formation in its N-terminal region, which binds to KIX in a predominantly folded conformation. The C-terminal region of c-Myb exhibits a much smaller helical propensity and likely folds via an induced-fit process after binding to KIX. We propose that the intrinsic secondary structure propensities of pKID and c-Myb determine their binding mechanisms, consistent with their functions as inducible and constitutive transcriptional activators.

Flexible and rigid structures in HIV-1 p17 matrix protein monitored by relaxation and amide proton exchange with NMR.

The HIV-1 p17 matrix protein is a multifunctional protein that interacts with other molecules including proteins and membranes. The dynamic structure between its folded and partially unfolded states can be critical for the recognition of interacting molecules. One of the most important roles of the p17 matrix protein is its localization to the plasma membrane with the Gag polyprotein. The myristyl group attached to the N-terminus on the p17 matrix protein functions as an anchor for binding to the plasma membrane. Biochemical studies revealed that two regions are important for its function: D14-L31 and V84-V88. Here, the dynamic structures of the p17 matrix protein were studied using NMR for relaxation and amide proton exchange experiments at the physiological pH of 7.0. The results revealed that the α12-loop, which includes the 14-31 region, was relatively flexible, and that helix 4, including the 84-88 region, was the most protected helix in this protein. However, the residues in the α34-loop near helix 4 had a low order parameter and high exchange rate of amide protons, indicating high flexibility. This region is probably flexible because this loop functions as a hinge for optimizing the interactions between helices 3 and 4. The C-terminal long region of K113-Y132 adopted a disordered structure. Furthermore, the C-terminal helix 5 appeared to be slightly destabilized due to the flexible C-terminal tail based on the order parameters. Thus, the dynamic structure of the p17 matrix protein may be related to its multiple functions.

Dynamics and interactions of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are widespread in eukaryotes and participate in a variety of important cellular processes. Numerous studies using state-of-the-art experimental and theoretical methods have advanced our understanding of IDPs and revealed that disordered regions engage in a large repertoire of intra- and intermolecular interactions through their conformational dynamics, thereby regulating many intracellular functions in concert with folded domains. The mechanisms by which IDPs interact with their partners are diverse, depending on their conformational propensities, and include induced fit, conformational selection, and their mixtures. In addition, IDPs are implicated in many diseases, and progress has been made in designing inhibitors of IDP-mediated interactions. Here we review these recent advances with a focus on the dynamics and interactions of IDPs.

Crucial Residue for Tuning Thermal Relaxation Kinetics in the Biliverdin-binding Cyanobacteriochrome Photoreceptor Revealed by Site-saturation Mutagenesis.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to the phytochromes sensing red and far-red light reversibly. Only the cGMP phosphodiesterase/Adenylate cyclase/FhlA (GAF) domain is needed for chromophore incorporation and proper photoconversion. The CBCR GAF domains covalently ligate linear tetrapyrrole chromophores and show reversible photoconversion between two light-absorbing states. In most cases, the two light-absorbing states are stable under dark conditions, but in some cases, the photoproduct state undergoes thermal relaxation back to the dark-adapted state during thermal relaxation. In this study, we examined the engineered CBCR GAF domain, AnPixJg2_BV4. AnPixJg2_BV4 covalently binds biliverdin IX-alpha (BV) and shows reversible photoconversion between a far-red-absorbing Pfr dark-adapted state and an orange-absorbing Po photoproduct state. Because the BV is an intrinsic chromophore of mammalian cells and absorbs far-red light penetrating into deep tissues, BV-binding CBCR molecules are useful for the development of optogenetic and bioimaging tools used in mammals. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis on the Phe319 position. We succeeded in obtaining variant molecules with higher chromophore-binding efficiency and higher molar extinction coefficient. Furthermore, we observed a wide variation in thermal relaxation kinetics, with an 81-fold difference between the slowest and fastest rates. Both molecules with relatively slow and fast thermal relaxation would be advantageous for optogenetic control.

Reentrant condensation of a multicomponent cola/milk system induced by polyphosphate.

Reentrant condensation (RC) is a protein behavior in which the protein solution shifts between the one- and two-phase state more than twice by increasing a single parameter. Although RC would be a candidate mechanism for the physicochemical design of food additives, no realistic model has been established under diverse contaminants like food materials. Here, we found that a mixture of cola and milk yielded RC. At pH 3.2-3.6, cola induced milk condensation at 30-40%, while lower or higher concentrations of cola did not. Furthermore, we reduced this cola/milk system to two pure components, casein in milk and polyphosphate (polyP) in cola, and investigated the characteristics of casein concentration and zeta potential. This was the first experimental demonstration of RC occurrence in a multicomponent system. The well-characterized cola/milk system would explore both the universal nature of proteins and the industrial application of RC.

Graded enhancement of p53 binding to CREB-binding protein (CBP) by multisite phosphorylation.

The transcriptional activity of p53 is regulated by a cascade of posttranslational modifications. Although acetylation of p53 by CREB-binding protein (CBP)/p300 is known to be indispensable for p53 activation, the role of phosphorylation, and in particular multisite phosphorylation, in activation of CBP/p300-dependent p53 transcriptional pathways remains unclear. We investigated the role of single site and multiple site phosphorylation of the p53 transactivation domain in mediating its interaction with CBP and with the ubiquitin ligase HDM2. Phosphorylation at Thr18 functions as an on/off switch to regulate binding to the N-terminal domain of HDM2. In contrast, binding to CBP is modulated by the extent of p53 phosphorylation; addition of successive phosphoryl groups enhances the affinity for the TAZ1, TAZ2, and KIX domains of CBP in an additive manner. Activation of p53-dependent transcriptional pathways requires that p53 compete with numerous cellular transcription factors for binding to limiting amounts of CBP/p300. Multisite phosphorylation represents a mechanism for a graded p53 response, with each successive phosphorylation event resulting in increasingly efficient recruitment of CBP/p300 to p53-regulated transcriptional programs, in the face of competition from cellular transcription factors. Multisite phosphorylation thus acts as a rheostat to enhance binding to CBP/p300 and provides a plausible mechanistic explanation for the gradually increasing p53 response observed following prolonged or severe genotoxic stress.

Accurate prediction of protein folding mechanisms by simple structure-based statistical mechanical models.

Recent breakthroughs in highly accurate protein structure prediction using deep neural networks have made considerable progress in solving the structure prediction component of the 'protein folding problem'. However, predicting detailed mechanisms of how proteins fold into specific native structures remains challenging, especially for multidomain proteins constituting most of the proteomes. Here, we develop a simple structure-based statistical mechanical model that introduces nonlocal interactions driving the folding of multidomain proteins. Our model successfully predicts protein folding processes consistent with experiments, without the limitations of protein size and shape. Furthermore, slight modifications of the model allow prediction of disulfide-oxidative and disulfide-intact protein folding. These predictions depict details of the folding processes beyond reproducing experimental results and provide a rationale for the folding mechanisms. Thus, our physics-based models enable accurate prediction of protein folding mechanisms with low computational complexity, paving the way for solving the folding process component of the 'protein folding problem'.

Rational peptide design for inhibition of the KIX-MLL interaction.

The kinase-inducible domain interacting (KIX) domain is an integral part of the general transcriptional coactivator CREB-binding protein, and has been associated with leukemia, cancer, and various viral diseases. Hence, the KIX domain has attracted considerable attention in drug discovery and development. Here, we rationally designed a KIX inhibitor using a peptide fragment corresponding to the transactivation domain (TAD) of the transcriptional activator, mixed-lineage leukemia protein (MLL). We performed theoretical saturation mutagenesis using the Rosetta software to search for mutants expected to bind KIX more tightly than the wild-type MLL TAD. Mutant peptides with higher helical propensities were selected for experimental characterization. We found that the T2857W mutant of the MLL TAD peptide had the highest binding affinity for KIX compared to the other 12 peptides designed in this study. Moreover, the peptide had a high inhibitory effect on the KIX-MLL interaction with a half-maximal inhibitory concentration close to the dissociation constant for this interaction. To our knowledge, this peptide has the highest affinity for KIX among all previously reported inhibitors that target the MLL site of KIX. Thus, our approach may be useful for rationally developing helical peptides that inhibit protein-protein interactions implicated in the progression of various diseases.

Introduction of reversible cysteine ligation ability to the biliverdin-binding cyanobacteriochrome photoreceptor.

Cyanobacteriochrome (CBCR) photoreceptors are distantly related to the canonical red/far-red reversible phytochrome photoreceptors. In the case of the CBCRs, only the GAF domain is required for chromophore incorporation and photoconversion. The GAF domains of CBCR are highly diversified into many lineages to sense various colors of light. These CBCR GAF domains are divided into two types: those possessing only the canonical Cys residue and those with both canonical and second Cys residues. The canonical Cys residue stably ligates to the chromophore in both cases. The second Cys residue mostly shows reversible adduct formation with the chromophore during photoconversion for spectral tuning. In this study, we focused on the CBCR GAF domain AnPixJg2_BV4, which possesses only the canonical Cys residue. AnPixJg2_BV4 covalently ligates to the biliverdin (BV) chromophore and shows far-red/orange reversible photoconversion. Because BV is a mammalian intrinsic chromophore, BV-binding molecules are advantageous for in vivo optogenetic and bioimaging tool development. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis and serendipitously obtained a unique variant molecule that showed far-red/blue reversible photoconversion, in which the Cys residue was introduced near the chromophore. This introduced Cys residue functioned as the second Cys residue that reversibly ligated with the chromophore. Because the position of the introduced Cys residue is distinct from the known second Cys residues, the variant molecule obtained in this study would expand our knowledge about the spectral tuning mechanism of CBCRs and contribute to tool development.

Quantitative analysis of multisite protein-ligand interactions by NMR: binding of intrinsically disordered p53 transactivation subdomains with the TAZ2 domain of CBP.

Determination of affinities and binding sites involved in protein-ligand interactions is essential for understanding molecular mechanisms in biological systems. Here we combine singular value decomposition and global analysis of NMR chemical shift perturbations caused by protein-protein interactions to determine the number and location of binding sites on the protein surface and to measure the binding affinities. Using this method we show that the isolated AD1 and AD2 binding motifs, derived from the intrinsically disordered N-terminal transactivation domain of the tumor suppressor p53, both interact with the TAZ2 domain of the transcriptional coactivator CBP at two binding sites. Simulations of titration curves and line shapes show that a primary dissociation constant as small as 1-10 nM can be accurately estimated by NMR titration methods, provided that the primary and secondary binding processes are coupled. Unexpectedly, the site of binding of AD2 on the hydrophobic surface of TAZ2 overlaps with the binding site for AD1, but AD2 binds TAZ2 more tightly. The results highlight the complexity of interactions between intrinsically disordered proteins and their targets. Furthermore, the association rate of AD2 to TAZ2 is estimated to be 1.7 × 10(10) M(-1) s(-1), approaching the diffusion-controlled limit and indicating that intrinsic disorder plus complementary electrostatics can significantly accelerate protein binding interactions.