The intrinsic substrate specificity of the human tyrosine kinome.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

The feasibility of determining kinetic constants from isothermal titration calorimetry data.

Isothermal titration calorimetry (ITC) has long been established as an excellent means to determine the thermodynamic parameters of biomolecular interactions. More recently, efforts have focused on exploiting the power/time trace (the "thermogram") resulting from ITC experiments to glean kinetic association and dissociation rates for these interactions. The success of such analyses rests on the ability of algorithms to simulate with high accuracy the output of the calorimeter. Thus, several critical factors must be taken into account: the injection protocol, the kinetics of the interaction, accurate discovery of the instrumental response to heat signals, and the addition of unrelated signals. All of these aspects of extracting kinetic constants from thermograms have been considered and addressed in the current work. To validate the resultant methods, we performed several ITC experiments, titrating small-molecule inhibitors into solutions of bovine carbonic anhydrase II or titrating lysozyme into solutions of anti-lysozyme nanobodies. We found that our methods could arrive at kinetic constants that were close to the known values for these interactions taken from other methods. Finally, the effort to improve ITC kinetic characterizations uncovered a set of best practices for both the calorimetric experiment and the subsequent analyses (termed "kinetically optimized ITC" or "KO-ITC") that is detailed in this work.

A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA.

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.

Fast and Robust Quantification of Detergent Micellization Thermodynamics from Isothermal Titration Calorimetry.

Detergents are widely used in modern in vitro biochemistry and biophysics, in particular to aid the characterization of integral membrane proteins. An important characteristic of these chemicals in aqueous solutions is the concentration above which their molecular monomers self-associate to form micelles, termed the critical micellar concentration (CMC). Micelles are supramolecular assemblies arranged with the hydrophobic portions oriented inward and the hydrophilic head groups positioned outward to interact with the aqueous solvent. Knowledge of the CMC is not only of practical relevance but also of theoretical interest because it provides thermodynamic insights. Isothermal titration calorimetry (ITC) is a powerful method to determine CMCs, as it furnishes additional information on the enthalpy and entropy of micellization. Here we describe our extension of previous methods to determine CMCs and other thermodynamic parameters from ITC demicellization curves. The new algorithm, incorporated into the stand-alone software package D/STAIN, analyzes ITC demicellization curves by taking advantage of state-of-the-art thermogram-integration techniques and automatically providing rigorous confidence intervals on the refined parameters. As a demonstration of the software's capabilities, we undertook ITC experiments to determine the respective CMCs of n-octyl ß-d-glucopyranoside (OG), n-dodecyl ß-d-maltopyranoside (DDM), and lauryldimethylamine N-oxide (LDAO). Motivated by the fact that in vitro membrane protein studies often require additives such as precipitants (e.g., polyethylene glycol (PEG)), we also carried out ITC demicellization studies in the presence of PEG3350, finding in all cases that PEG had significant effects on the thermodynamics of detergent micellization.

Using modern approaches to sedimentation velocity to detect conformational changes in proteins.

It has been known for decades that proteins undergo conformational changes in response to binding ligands. Such changes are usually accompanied by a loss of entropy by the protein, and thus conformational changes are integral to the thermodynamics of ligand association. Methods to detect these alterations are numerous; here, we focus on the sedimentation velocity (SV) mode of AUC, which has several advantages, including ease of use and rigorous data-selection criteria. In SV, it is assumed that conformational changes manifest primarily as differences in the sedimentation coefficient (the s-value). Two methods of determining s-value differences were assessed. The first method used the widely adopted c(s) distribution to gather statistics on the s-value differences to determine whether the observed changes were reliable. In the second method, a decades-old technique called "difference SV" was revived and updated to address its viability in this era of modern instrumentation. Both methods worked well to determine the extent of conformational changes to three model systems. Both simulations and experiments were used to explore the strengths and limitations of the methods. Finally, software incorporating these methodologies was produced.

Using two-site binding models to analyze microscale thermophoresis data.

The emergence of microscale thermophoresis (MST) as a technique for determining the dissociation constants for bimolecular interactions has enabled these quantities to be measured in systems that were previously difficult or impracticable. However, most models for analyses of these data featured the assumption of a simple 1:1 binding interaction. The only model widely used for multiple binding sites was the Hill equation. Here, we describe two new MST analytic models that assume a 1:2 binding scheme: the first features two microscopic binding constants (KD(1) and KD(2)), while the other assumes symmetry in the bivalent molecule, culminating in a model with a single macroscopic dissociation constant (KD,M) and a single factor (α) that accounts for apparent cooperativity in the binding. We also discuss the general applicability of the Hill equation for MST data. The performances of the algorithms on both real and simulated data are assessed, and implementation of the algorithms in the MST analysis program PALMIST is discussed.

Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3 mutation.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. An X-linked form of CMT (CMTX6) is caused by a missense mutation (R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. PDK3 is one of 4 isoenzymes that negatively regulate the activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation of its first catalytic component pyruvate dehydrogenase (designated as E1). Mitochondrial PDC catalyses the oxidative decarboxylation of pyruvate to acetyl CoA and links glycolysis to the energy-producing Krebs cycle. We have previously shown the R158H mutation confers PDK3 enzyme hyperactivity. In this study we demonstrate that the increased PDK3 activity in patient fibroblasts (PDK3(R158H)) leads to the attenuation of PDC through hyper-phosphorylation of E1 at selected serine residues. This hyper-phosphorylation can be reversed by treating the PDK3(R158H) fibroblasts with the PDK inhibitor dichloroacetate (DCA). In the patient cells, down-regulation of PDC leads to increased lactate, decreased ATP and alteration of the mitochondrial network. Our findings highlight the potential to develop specific drug targeting of the mutant PDK3 as a therapeutic approach to treating CMTX6.

Structure-based design and mechanisms of allosteric inhibitors for mitochondrial branched-chain α-ketoacid dehydrogenase kinase.

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, (S)-α-chloro-phenylpropionic acid [(S)-CPP]. Crystal structures of the BDK-(S)-CPP complex show that (S)-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of (S)-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure.

Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket.

Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 µM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes.

Benzothiophene carboxylate derivatives as novel allosteric inhibitors of branched-chain α-ketoacid dehydrogenase kinase.

The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation.BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6- dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC(50) = 3.19 µM). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-α-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T(1/2) = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[ b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[ b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations.

A new locus for X-linked dominant Charcot-Marie-Tooth disease (CMTX6) is caused by mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene.

Hereditary motor and sensory disorders of the peripheral nerve form one of the most common groups of human genetic diseases collectively called Charcot-Marie-Tooth (CMT) neuropathy. Using linkage analysis in a three generation kindred, we have mapped a new locus for X-linked dominant CMT to chromosome Xp22.11. A microsatellite scan of the X chromosome established significant linkage to several markers including DXS993 (Zmax = 3.16; θ = 0.05). Extended haplotype analysis refined the linkage region to a 1.43-Mb interval flanked by markers DXS7110 and DXS8027. Whole exome sequencing identified a missense mutation c.G473A (p.R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. The change localized within the 1.43-Mb linkage interval, segregated with the affected phenotype and was excluded in ethnically matched control chromosomes. PDK3 is one of the four isoenzymes regulating the pyruvate dehydrogenase complex (PDC), by reversible phosphorylation, and is a nuclear-coded protein located in the mitochondrial matrix. PDC catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA and is a key enzyme linking glycolysis to the energy-producing Krebs cycle and lipogenic pathways. We found that the R158H mutation confers enzyme hyperactivity and binds with stronger affinity than the wild-type to the inner-lipoyl (L2) domain of the E2p chain of PDC. Our findings suggest a reduced pyruvate flux due to R158H mutant PDK3-mediated hyper-phosphorylation of the PDC as the underlying pathogenic cause of peripheral neuropathy. The results highlight an important causative link between peripheral nerve degeneration and an essential bioenergetic or biosynthetic pathway required for the maintenance of peripheral nerves.

Biophysical and biochemical studies support TP0094 as a phosphotransacetylase in an acetogenic energy-conservation pathway in Treponema pallidum.

The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism's energy needs likely exceed the modest output from glycolysis alone. Recently, building on our structure-function studies of T. pallidum lipoproteins, we proposed a "flavin-centric" metabolic lifestyle for the organism that partially resolves this conundrum. As a part of the hypothesis, we have proposed that T. pallidum contains an acetogenic energy-conservation pathway that catabolizes D-lactate, yielding acetate, reducing equivalents for the generation and maintenance of chemiosmotic potential, and ATP. We already have confirmed the D-lactate dehydrogenase activity in T. pallidum necessary for this pathway to operate. In the current study, we focused on another enzyme ostensibly involved in treponemal acetogenesis, phosphotransacetylase (Pta). This enzyme is putatively identified as TP0094 and, in this study, we determined a high-resolution (1.95 Å) X-ray crystal structure of the protein, finding that its fold comports with other known Pta enzymes. Further studies on its solution behavior and enzyme activity confirmed that it has the properties of a Pta. These results are consistent with the proposed acetogenesis pathway in T. pallidum, and we propose that the protein be referred to henceforth as TpPta.

Measuring the KD of Protein-Ligand Interactions Using Microscale Thermophoresis.

Microscale thermophoresis (MST) has become a widely used technique to determine the KD or EC50 of protein-ligand interactions. The method exploits the tendency of macromolecules to migrate along a thermal gradient (i.e., thermophoresis). Differences in thermophoresis as a function of the liganded state of a macromolecule can be measured and assembled into a binding curve that can be analyzed to yield KD. In this protocol, we outline a simple experiment designed for new MST users, with the goal of using readily available, inexpensive materials to plan, execute, and analyze an MST experiment.

Effect of subunit IV on superoxide generation by Rhodobacter sphaeroides cytochrome bc(1) complex.

Previous studies indicate that the three-subunit cytochrome bc(1) core complex of Rhodobacter sphaeroides contains a fraction of the electron transfer activity of the wild-type enzyme. Addition of subunit IV to the core complex increases electron transfer activity to the same level as that of the wild-type complex. This activity increase may result from subunit IV preventing electron leakage, from the low potential electron transfer chain, and reaction with molecular oxygen, producing superoxide anion. This suggestion is based on the following observations: (1) the extent of cytochrome b reduction in the three-subunit core complex, by ubiquinol, in the presence of antimycin A, never reaches the same level as that in the wild-type complex; (2) the core complex produces 4 times as much superoxide anion as does the wild-type complex; and (3) when the core complex is reconstituted with subunit IVs having varying reconstitutive activities, the activity increase in reconstituted complexes correlates with superoxide production decrease and extent of cytochrome b reduction increase.

Structural basis for inactivation of the human pyruvate dehydrogenase complex by phosphorylation: role of disordered phosphorylation loops.

We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-alpha (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-alpha prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-alpha, which nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p, negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.

Biophysical insights into a highly selective l-arginine-binding lipoprotein of a pathogenic treponeme.

Biophysical and biochemical studies on the lipoproteins and other periplasmic proteins from the spirochetal species Treponema pallidum have yielded numerous insights into the functioning of the organism's peculiar membrane organization, its nutritional requirements, and intermediary metabolism. However, not all T. pallidum proteins have proven to be amenable to biophysical studies. One such recalcitrant protein is Tp0309, a putative polar-amino-acid-binding protein of an ABC transporter system. To gain further information on its possible function, a homolog of the protein from the related species T. vincentii was used as a surrogate. This protein, Tv2483, was crystallized, resulting in the determination of its crystal structure at a resolution of 1.75 Å. The protein has a typical fold for a ligand-binding protein, and a single molecule of l-arginine was bound between its two lobes. Differential scanning fluorimetry and isothermal titration calorimetry experiments confirmed that l-arginine bound to the protein with unusually high selectivity. However, further comparison to Tp0309 showed differences in key amino-acid-binding residues may impart an alternate specificity for the T. pallidum protein.

An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue.

Microtubules are heavily regulated dynamic polymers of αß-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the cytoplasmic linker associated protein (CLASP) family that reduce catastrophe and/or increase rescue also contain arrayed TOGs, but how CLASP TOGs contribute to activity is poorly understood. Here, using Saccharomyces cerevisiae Stu1 as a model CLASP, we report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. The two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. The structure of Stu1-TOG2 reveals a CLASP-specific residue that likely confers distinctive tubulin-binding properties. The isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro, contradicting the expectation that regulatory activity requires an array of TOGs. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function.

Targeting hepatic pyruvate dehydrogenase kinases restores insulin signaling and mitigates ChREBP-mediated lipogenesis in diet-induced obese mice.

OBJECTIVE: Mitochondrial pyruvate dehydrogenase kinases 1-4 (PDKs1-4) negatively regulate activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation. PDKs play a pivotal role in maintaining energy homeostasis and contribute to metabolic flexibility by attenuating PDC activity in various mammalian tissues. Cumulative evidence has shown that the up-regulation of PDK4 expression is tightly associated with obesity and diabetes. In this investigation, we test the central hypothesis that PDKs1-4 are a pharmacological target for lowering glucose levels and restoring insulin sensitivity in obesity and type 2 diabetes (T2D). METHODS: Diet-induced obese (DIO) mice were treated with a liver-specific pan-PDK inhibitor 2-[(2,4-dihydroxyphenyl) sulfonyl]isoindoline-4,6-diol (PS10) for four weeks, and results compared with PDK2/PDK4 double knockout (DKO) mice on the same high fat diet (HFD). RESULTS: Both PS10-treated DIO mice and HFD-fed DKO mice showed significantly improved glucose, insulin and pyruvate tolerance, compared to DIO controls, with lower plasma insulin levels and increased insulin signaling in liver. In response to lower glucose levels, phosphorylated AMPK in PS10-treated DIO and HFD-fed DKO mice is upregulated, accompanied by decreased nuclear carbohydrate-responsive element binding protein (ChREBP). The reduced ChREBP signaling correlates with down-regulation of hepatic lipogenic enzymes (ACC1, FAS, and SCD1), leading to markedly diminished hepatic steatosis in both study groups, with lower circulating cholesterol and triacylglyceride levels as well as reduced fat mass. PS10-treated DIO as well as DKO mice showed predominant fatty acid over glucose oxidation. However, unlike systemic DKO mice, increased hepatic PDC activity alone in PS10-treated DIO mice does not raise the plasma total ketone body level. CONCLUSION: Our findings establish that specific targeting of hepatic PDKs with the PDK inhibitor PS10 is an effective therapeutic approach to maintaining glucose and lipid homeostasis in obesity and T2D, without the harmful ketoacidosis associated with systemic inhibition of PDKs.

The BCKDH Kinase and Phosphatase Integrate BCAA and Lipid Metabolism via Regulation of ATP-Citrate Lyase.

Branched-chain amino acids (BCAA) are strongly associated with dysregulated glucose and lipid metabolism, but the underlying mechanisms are poorly understood. We report that inhibition of the kinase (BDK) or overexpression of the phosphatase (PPM1K) that regulates branched-chain ketoacid dehydrogenase (BCKDH), the committed step of BCAA catabolism, lowers circulating BCAA, reduces hepatic steatosis, and improves glucose tolerance in the absence of weight loss in Zucker fatty rats. Phosphoproteomics analysis identified ATP-citrate lyase (ACL) as an alternate substrate of BDK and PPM1K. Hepatic overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis. BDK and PPM1K transcript levels were increased and repressed, respectively, in response to fructose feeding or expression of the ChREBP-ß transcription factor. These studies identify BDK and PPM1K as a ChREBP-regulated node that integrates BCAA and lipid metabolism. Moreover, manipulation of the BDK:PPM1K ratio relieves key metabolic disease phenotypes in a genetic model of severe obesity.

Identification of amino acid residues essential for reconstitutive activity of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides.

A region of subunit IV comprising residues 77-85 is identified as essential for interaction with the core complex to restore the bc(1) activity (reconstitutive activity). Recombinant subunit IV mutants with single or multiple alanine substitution at this region were generated and characterized to identify the essential amino acid residues. Residues 81-84, with sequence of YRYR, are required for reconstitutive activity of subunit IV, because a mutant with these four residues substituted with alanine has little activity, while a mutant with alanine substitution at residues 77-80 and 85 have the same reconstitutive activity as that of the wild-type IV. The positively charged group at R-82 and R-84 and both the hydroxyl group and aromatic group at Y-81 and Y-83 are essential. The interactions between these four residues of subunit IV and residues of core subunits are also responsible for the stability of the complex. However, these interactions are not essential for the incorporation of subunit IV into the bc(1) complex.