Validation of aminodeoxychorismate synthase and anthranilate synthase as novel targets for bispecific antibiotics inhibiting conserved protein-protein interactions.

Multi-resistant bacteria are a rapidly emerging threat to modern medicine. It is thus essential to identify and validate novel antibacterial targets that promise high robustness against resistance-mediating mutations. This can be achieved by simultaneously targeting several conserved function-determining protein-protein interactions in enzyme complexes from prokaryotic primary metabolism. Here, we selected two evolutionary related glutamine amidotransferase complexes, aminodeoxychorismate synthase and anthranilate synthase, that are required for the biosynthesis of folate and tryptophan in most prokaryotic organisms. Both enzymes rely on the interplay of a glutaminase and a synthase subunit that is conferred by a highly conserved subunit interface. Consequently, inhibiting subunit association in both enzymes by one competing bispecific inhibitor has the potential to suppress bacterial proliferation. We comprehensively verified two conserved interface hot-spot residues as potential inhibitor-binding sites in vitro by demonstrating their crucial role in subunit association and enzymatic activity. For in vivo target validation, we generated genomically modified Escherichia coli strains in which subunit association was disrupted by modifying these central interface residues. The growth of such strains was drastically retarded on liquid and solid minimal medium due to a lack of folate and tryptophan. Remarkably, the bacteriostatic effect was observed even in the presence of heat-inactivated human plasma, demonstrating that accessible host metabolite concentrations do not compensate for the lack of folate and tryptophan within the tested bacterial cells. We conclude that a potential inhibitor targeting both enzyme complexes will be effective against a broad spectrum of pathogens and offer increased resilience against antibiotic resistance. IMPORTANCE: Antibiotics are indispensable for the treatment of bacterial infections in human and veterinary medicine and are thus a major pillar of modern medicine. However, the exposure of bacteria to antibiotics generates an unintentional selective pressure on bacterial assemblies that over time promotes the development or acquisition of resistance mechanisms, allowing pathogens to escape the treatment. In that manner, humanity is in an ever-lasting race with pathogens to come up with new treatment options before resistances emerge. In general, antibiotics with novel modes of action require more complex pathogen adaptations as compared to chemical derivates of existing entities, thus delaying the emergence of resistance. In this contribution, we use modified Escherichia coli strains to validate two novel targets required for folate and tryptophan biosynthesis that can potentially be targeted by one and the same bispecific protein-protein interaction inhibitor and promise increased robustness against bacterial resistances.

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase.

The artificial regulation of enzymatic activity by light is an important goal of synthetic biology that can be achieved by the incorporation of light-responsive noncanonical amino acids via genetic code expansion. Here, we apply this concept to anthranilate synthase from Salmonella typhimurium (stTrpE). This enzyme catalyzes the first step of tryptophan biosynthesis, and its activity is feedback-inhibited by the binding of the end-product of the pathway to an allosteric site. To put this feedback inhibition of stTrpE by tryptophan under the control of light, we individually replaced 15 different amino acid residues with the photosensitive noncanonical amino acid o-nitrobenzyl-O-tyrosine (ONBY). ONBY contains a sterically demanding caging group that was meant to cover the allosteric site. Steady-state enzyme kinetics showed that the negative effect of tryptophan on the catalytic activity of the two variants stTrpE-K50ONBY and stTrpE-Y455ONBY was diminished compared to the wild-type enzyme by 1 to 2 orders of magnitude. Upon light-induced decaging of ONBY to the less space-consuming tyrosine residue, tryptophan binding to the allosteric site was restored and catalytic activity was inhibited almost as efficiently as observed for wild-type stTrpE. Based on these results, direct photocontrol of feedback inhibition of stTrpE-K50ONBY and stTrpE-Y455ONBY could be achieved by irradiation during the reaction. Molecular modeling studies allowed us to rationalize the observed functional conversion from the noninhibited caged to the tryptophan-inhibited decaged states. Our study shows that feedback inhibition, which is an important mechanism to regulate key metabolic enzymes, can be efficiently controlled by the purposeful use of light-responsive noncanonical amino acids.

Reprogramming the Specificity of a Protein Interface by Computational and Data-Driven Design.

The formation of specific protein complexes in a cell is a non-trivial problem given the co-existence of thousands of different polypeptide chains. A particularly difficult case are two glutamine amidotransferase complexes (anthranilate synthase [AS] and aminodeoxychorismate synthase [ADCS]), which are composed of homologous pairs of synthase and glutaminase subunits. We have attempted to identify discriminating interface residues of the glutaminase subunit TrpG from AS, which are responsible for its specific interaction with the synthase subunit TrpEx and prevent binding to the closely related synthase subunit PabB from ADCS. For this purpose, TrpG-specific interface residues were grafted into the glutaminase subunit PabA from ADCS by two different approaches, namely a computational and a data-driven one. Both approaches resulted in PabA variants that bound TrpEx with higher affinity than PabB. Hence, we have accomplished a reprogramming of protein-protein interaction specificity that provides insights into the evolutionary adaptation of protein interfaces.

Prediction of quaternary structure by analysis of hot spot residues in protein-protein interfaces: the case of anthranilate phosphoribosyltransferases.

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic "hot spot" amino acids in the protein-protein interface that were predicted to be essential for self-association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer-dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures.

Relationship of Catalysis and Active Site Loop Dynamics in the (ßα)8-Barrel Enzyme Indole-3-glycerol Phosphate Synthase.

It is important to understand how the catalytic activity of enzymes is related to their conformational flexibility. We have studied this activity-flexibility correlation using the example of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (ssIGPS), which catalyzes the fifth step in the biosynthesis of tryptophan. ssIGPS is a thermostable representative of enzymes with the frequently encountered and catalytically versatile (ßα)8-barrel fold. Four variants of ssIGPS with increased catalytic turnover numbers were analyzed by transient kinetics at 25 °C, and wild-type ssIGPS was likewise analyzed both at 25 °C and at 60 °C. Global fitting with a minimal three-step model provided the individual rate constants for substrate binding, chemical transformation, and product release. The results showed that in both cases, namely, the application of activating mutations and temperature increase, the net increase in the catalytic turnover number is afforded by acceleration of the product release rate relative to the chemical transformation steps. Measurements of the solvent viscosity effect at 25 °C versus 60 °C confirmed this change in the rate-determining step with temperature, which is in accordance with a kink in the Arrhenius diagram of ssIGPS at ∼40 °C. When rotational diffusion rates of electron paramagnetic spin-labels attached to active site loop ß1α1 are plotted in the form of an Arrhenius diagram, kinks are observed at the same temperature. These findings, together with molecular dynamics simulations, demonstrate that a different degree of loop mobility correlates with different rate-limiting steps in the catalytic mechanism of ssIGPS.

Ancestral Tryptophan Synthase Reveals Functional Sophistication of Primordial Enzyme Complexes.

Modern enzyme complexes are characterized by a high catalytic efficiency and allosteric communication between the constituting protein subunits. We were interested in whether primordial enzyme complexes from extinct species displayed a similar degree of functional sophistication. To this end, we used ancestral sequence reconstruction to resurrect the α and ß subunits of the tryptophan synthase (TS) complex from the last bacterial common ancestor (LBCA), which presumably existed more than 3.4 billion years ago. We show that the LBCA TS subunits are thermostable and exhibit high catalytic activity. Moreover, they form a complex with αßßα stoichiometry whose crystal structure is similar to that of modern TS. Kinetic analysis revealed that the reaction intermediate indole is channeled from the α to the ß subunits and suggests that allosteric communication already occurred in LBCA TS.

Quantitative analysis of receptor tyrosine kinase-effector coupling at functionally relevant stimulus levels.

A major goal of current signaling research is to develop a quantitative understanding of how receptor activation is coupled to downstream signaling events and to functional cellular responses. Here, we measure how activation of the RET receptor tyrosine kinase on mouse neuroblastoma cells by the neurotrophin artemin (ART) is quantitatively coupled to key downstream effectors. We show that the efficiency of RET coupling to ERK and Akt depends strongly on ART concentration, and it is highest at the low (∼100 pM) ART levels required for neurite outgrowth. Quantitative discrimination between ERK and Akt pathway signaling similarly is highest at this low ART concentration. Stimulation of the cells with 100 pM ART activated RET at the rate of ∼10 molecules/cell/min, leading at 5-10 min to a transient peak of ∼150 phospho-ERK (pERK) molecules and ∼50 pAkt molecules per pRET, after which time the levels of these two signaling effectors fell by 25-50% while the pRET levels continued to slowly rise. Kinetic experiments showed that signaling effectors in different pathways respond to RET activation with different lag times, such that the balance of signal flux among the different pathways evolves over time. Our results illustrate that measurements using high, super-physiological growth factor levels can be misleading about quantitative features of receptor signaling. We propose a quantitative model describing how receptor-effector coupling efficiency links signal amplification to signal sensitization between receptor and effector, thereby providing insight into design principles underlying how receptors and their associated signaling machinery decode an extracellular signal to trigger a functional cellular outcome.

Evidence for the existence of elaborate enzyme complexes in the Paleoarchean era.

Due to the lack of macromolecular fossils, the enzymatic repertoire of extinct species has remained largely unknown to date. In an attempt to solve this problem, we have characterized a cyclase subunit (HisF) of the imidazole glycerol phosphate synthase (ImGP-S), which was reconstructed from the era of the last universal common ancestor of cellular organisms (LUCA). As observed for contemporary HisF proteins, the crystal structure of LUCA-HisF adopts the (ßα)8-barrel architecture, one of the most ancient folds. Moreover, LUCA-HisF (i) resembles extant HisF proteins with regard to internal 2-fold symmetry, active site residues, and a stabilizing salt bridge cluster, (ii) is thermostable and shows a folding mechanism similar to that of contemporary (ßα)8-barrel enzymes, (iii) displays high catalytic activity, and (iv) forms a stable and functional complex with the glutaminase subunit (HisH) of an extant ImGP-S. Furthermore, we show that LUCA-HisF binds to a reconstructed LUCA-HisH protein with high affinity. Our findings suggest that the evolution of highly efficient enzymes and enzyme complexes has already been completed in the LUCA era, which means that sophisticated catalytic concepts such as substrate channeling and allosteric communication existed already 3.5 billion years ago.

Molecular engineering of organophosphate hydrolysis activity from a weak promiscuous lactonase template.

Rapid evolution of enzymes provides unique molecular insights into the remarkable adaptability of proteins and helps to elucidate the relationship between amino acid sequence, structure, and function. We interrogated the evolution of the phosphotriesterase from Pseudomonas diminuta (PdPTE), which hydrolyzes synthetic organophosphates with remarkable catalytic efficiency. PTE is thought to be an evolutionarily "young" enzyme, and it has been postulated that it has evolved from members of the phosphotriesterase-like lactonase (PLL) family that show promiscuous organophosphate-degrading activity. Starting from a weakly promiscuous PLL scaffold (Dr0930 from Deinococcus radiodurans ), we designed an extremely efficient organophosphate hydrolase (OPH) with broad substrate specificity using rational and random mutagenesis in combination with in vitro activity screening. The OPH activity for seven organophosphate substrates was simultaneously enhanced by up to 5 orders of magnitude, achieving absolute values of catalytic efficiencies up to 10(6) M(-1) s(-1). Structural and computational analyses identified the molecular basis for the enhanced OPH activity of the engineered PLL variants and demonstrated that OPH catalysis in PdPTE and the engineered PLL differ significantly in the mode of substrate binding.

Kinetic mechanism of indole-3-glycerol phosphate synthase.

The (ßα)(8)-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop ß1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate binding and catalysis by sIGPS.

Experimental assessment of the importance of amino acid positions identified by an entropy-based correlation analysis of multiple-sequence alignments.

The analysis of a multiple-sequence alignment (MSA) with correlation methods identifies pairs of residue positions whose occupation with amino acids changes in a concerted manner. It is plausible to assume that positions that are part of many such correlation pairs are important for protein function or stability. We have used the algorithm H2r to identify positions k in the MSAs of the enzymes anthranilate phosphoribosyl transferase (AnPRT) and indole-3-glycerol phosphate synthase (IGPS) that show a high conn(k) value, i.e., a large number of significant correlations in which k is involved. The importance of the identified residues was experimentally validated by performing mutagenesis studies with sAnPRT and sIGPS from the archaeon Sulfolobus solfataricus. For sAnPRT, five H2r mutant proteins were generated by replacing nonconserved residues with alanine or the prevalent residue of the MSA. As a control, five residues with conn(k) values of zero were chosen randomly and replaced with alanine. The catalytic activities and conformational stabilities of the H2r and control mutant proteins were analyzed by steady-state enzyme kinetics and thermal unfolding studies. Compared to wild-type sAnPRT, the catalytic efficiencies (k(cat)/K(M)) were largely unaltered. In contrast, the apparent thermal unfolding temperature (T(M)(app)) was lowered in most proteins. Remarkably, the strongest observed destabilization (ΔT(M)(app) = 14 °C) was caused by the V284A exchange, which pertains to the position with the highest correlation signal [conn(k) = 11]. For sIGPS, six H2r mutant and four control proteins with alanine exchanges were generated and characterized. The k(cat)/K(M) values of four H2r mutant proteins were reduced between 13- and 120-fold, and their T(M)(app) values were decreased by up to 5 °C. For the sIGPS control proteins, the observed activity and stability decreases were much less severe. Our findings demonstrate that positions with high conn(k) values have an increased probability of being important for enzyme function or stability.

Activation of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus by removal of magnesium inhibition and acceleration of product release .

Anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Sulfolobus solfataricus (ssAnPRT) is encoded by the sstrpD gene and catalyzes the reaction of anthranilate (AA) with a complex of Mg(2+) and 5'-phosphoribosyl-alpha1-pyrophosphate (Mg.PRPP) to N-(5'-phosphoribosyl)-anthranilate (PRA) and pyrophosphate (PP(i)) within tryptophan biosynthesis. The ssAnPRT enzyme is highly thermostable (half-life at 85 degrees C = 35 min) but only marginally active at ambient temperatures (turnover number at 37 degrees C = 0.33 s(-1)). To understand the reason for the poor catalytic proficiency of ssAnPRT, we have isolated from an sstrpD library the activated ssAnPRT-D83G + F149S double mutant by metabolic complementation of an auxotrophic Escherichia coli strain. Whereas the activity of purified wild-type ssAnPRT is strongly reduced in the presence of high concentrations of Mg(2+) ions, this inhibition is no longer observed in the double mutant and the ssAnPRT-D83G single mutant. The comparison of the crystal structures of activated and wild-type ssAnPRT shows that the D83G mutation alters the binding mode of the substrate Mg.PRPP. Analysis of PRPP and Mg(2+)-dependent enzymatic activity indicates that this leads to a decreased affinity for a second Mg(2+) ion and thus reduces the concentration of enzymes with the inhibitory Mg(2).PRPP complex bound to the active site. Moreover, the turnover number of the double mutant ssAnPRT-D83G + F149S is elevated 40-fold compared to the wild-type enzyme, which can be attributed to an accelerated release of the product PRA. This effect appears to be mainly caused by an increased conformational flexibility induced by the F149S mutation, a hypothesis which is supported by the reduced thermal stability of the ssAnPRT-F149S single mutant.

Coupling and dynamics of subunits in the hexameric AAA+ chaperone ClpB.

The bacterial AAA+ protein ClpB and its eukaryotic homologue Hsp104 ensure thermotolerance of their respective organisms by reactivating aggregated proteins in cooperation with the Hsp70/Hsp40 chaperone system. Like many members of the AAA+ superfamily, the ClpB protomers form ringlike homohexameric complexes. The mechanical energy necessary to disentangle protein aggregates is provided by ATP hydrolysis at the two nucleotide-binding domains of each monomer. Previous studies on ClpB and Hsp104 show a complex interplay of domains and subunits resulting in homotypic and heterotypic cooperativity. Using mutations in the Walker A and Walker B nucleotide-binding motifs in combination with mixing experiments we investigated the degree of inter-subunit coupling with respect to different aspects of the ClpB working cycle. We find that subunits are tightly coupled with regard to ATPase and chaperone activity, but no coupling can be observed for ADP binding. Comparison of the data with statistical calculations suggests that for double Walker mutants, approximately two in six subunits are sufficient to abolish chaperone and ATPase activity completely. In further experiments, we determined the dynamics of subunit reshuffling. Our results show that ClpB forms a very dynamic complex, reshuffling subunits on a timescale comparable to steady-state ATP hydrolysis. We propose that this could be a protection mechanism to prevent very stable aggregates from becoming suicide inhibitors for ClpB.

Quantitative analysis of the activation mechanism of the multicomponent growth-factor receptor Ret.

Cytokines and growth factors signal by modulating the interactions between multiple receptor components to form an activated receptor complex. The quantitative details of the activation mechanisms of this important class of receptors are not well understood. Using receptor phosphorylation measurements in live cells, as well as mathematical modeling and data fitting, we have characterized the multistep mechanism by which the GDNF-family neurotrophin artemin (ART), together with its co-receptor GDNF-family receptor alpha3 (GFRalpha3), brings about activation of the Ret receptor tyrosine kinase through formation of a pentameric signaling complex: ART-(GFRalpha3)(2)-(Ret)(2). By systematically varying the concentrations of ART and cell-surface GFRalpha3, we establish both the sequence of steps by which the signaling complex forms and the affinities of all the steps, including the two-dimensional affinities of the steps involving protein-protein interactions between membrane-bound species. Our results reveal the ways in which the individual binary interactions involved in the activation of a multicomponent receptor govern the receptor's functional properties.

Intrinsic inhibition of the Hsp90 ATPase activity.

The molecular chaperone Hsp90 is required for the folding and activation of a large number of substrate proteins. These are involved in essential cellular processes ranging from signal transduction to viral replication. For the activation of its substrates, Hsp90 binds and hydrolyzes ATP, which is the key driving force for conformational conversions within the dimeric chaperone. Dimerization of Hsp90 is mediated by a C-terminal dimerization site. In addition, there is a transient ATP-induced dimerization of the two N-terminal ATP-binding domains. The resulting ring-like structure is thought to be the ATPase-active conformation. Hsp90 is a slow ATPase with a turnover number of 1 ATP/min for the yeast protein. A key question for understanding the molecular mechanism of Hsp90 is how ATP hydrolysis is regulated and linked to conformational changes. In this study, we analyzed the activation process structurally and biochemically with a view to identify the conformational limitations of the ATPase reaction cycle. We showed that the first 24 amino acids stabilize the N-terminal domain in a rigid state. Their removal confers flexibility specifically to the region between amino acids 98 and 120. Most surprisingly, the deletion of this structure results in the complete loss of ATPase activity and in increased N-terminal dimerization. Complementation assays using heterodimeric Hsp90 show that this rigid lid acts as an intrinsic kinetic inhibitor of the Hsp90 ATPase cycle preventing N-terminal dimerization in the ground state. On the other hand, this structure acts, in concert with the 24 N-terminal amino acids of the other N-terminal domain, to form an activated ATPase and thus regulates the turnover number of Hsp90.

Biochemical coupling of the two nucleotide binding domains of ClpB: covalent linkage is not a prerequisite for chaperone activity.

ClpB cooperates with the DnaK chaperone system in the reactivation of protein from aggregates and is a member of the ATPases associated with a variety of cellular activities (AAA+) protein family. The underlying disaggregation reaction is dependent on ATP hydrolysis at both AAA cassettes of ClpB but the role of each AAA cassette in the reaction cycle is largely unknown. Here we analyze the activity of the separately expressed and purified nucleotide binding domains of ClpB from Thermus thermophilus. The two fragments show different biochemical properties: the first construct is inactive in ATPase activity assays and binds nucleotides weakly, the second construct has a very high ATPase activity and interacts tightly with nucleotides. Both individual fragments have lost their chaperone function and are not able to form large oligomers. When combined in solution, however, the two fragments form a stable heterodimer with oligomerization capacities equivalent to wild-type ClpB. This non-covalent complex regains activity in reactivating protein aggregates in cooperation with the DnaK chaperone system. Upon complex formation the ATPase activity of fragment 2 is reduced to a level similar to wild-type ClpB. Hence functional ClpB can be reassembled from its isolated AAA cassettes showing that covalent linkage of these domains is not a prerequisite for the chaperone activity. The observation that the intrinsically high ATPase activity of AAA2 is suppressed by AAA1 allows a hypothetical assignment of their mechanistic function. Whereas the energy gained upon ATP hydrolysis at the AAA2 is likely to drive a conformational change of the structure of ClpB, AAA1 might function as a regulator of the chaperone cycle.

A chaperone network for the resolubilization of protein aggregates: direct interaction of ClpB and DnaK.

The molecular chaperones ClpB (Hsp104) and DnaK (Hsp70) co-operate in the ATP-dependent resolubilization of aggregated proteins. A sequential mechanism has been proposed for this reaction; however, the mechanism and the functional interplay between both chaperones remain poorly defined. Here, we show for the first time that complex formation of ClpB and DnaK can be detected by using various types of affinity chromatography methods. The finding that the DnaK chaperone of Escherichia coli is not co-operating with ClpB from Thermus thermophilus further strengthens the specificity of this complex. The affinity of the complex is weak and interaction between both chaperones is nucleotide-dependent. The presence of ADP, which is shown to cause dissociation of ClpB(Tth), as well as ClpB deletion mutants incapable of oligomer formation prevent ClpB-DnaK complex formation. The experiments presented indicate a correlation between the oligomeric state of ClpB and its ability to interact with DnaK. The chaperone complex described here might facilitate transfer of intermediates between ClpB and DnaK during refolding of substrates from aggregates.

The N terminus of ClpB from Thermus thermophilus is not essential for the chaperone activity.

ClpB from Thermus thermophilus belongs to the Clp/Hsp100 protein family and reactivates protein aggregates in cooperation with the DnaK chaperone system. The mechanism of protein reactivation and interaction with the DnaK system remains unclear. ClpB possesses two nucleotide binding domains, which are essential for function and show a complex allosteric behavior. The role of the N-terminal domain that precedes the first nucleotide binding domain is largely unknown. We purified and characterized an N-terminal shortened ClpB variant (ClpBDeltaN; amino acids 140-854), which remained active in refolding assays with three different substrate proteins. In addition the N-terminal truncation did not significantly change the nucleotide binding affinities, the nucleotide-dependent oligomerization, and the allosteric behavior of the protein. In contrast casein binding and stimulation of the ATPase activity by kappa-casein were affected. These results suggest that the N-terminal domain is not essential for the chaperone function, does not influence the binding of nucleotides, and is not involved in the formation of intermolecular contacts. It contributes to the casein binding site of ClpB, but other substrate proteins do not necessarily interact with the N terminus. This indicates a substantial difference in the binding mode of kappa-casein that is often used as model substrate for ClpB and other possibly more suitable substrate proteins.