Modulation of the ATPase cycle of BiP by peptides and proteins

BiP, the Hsp70 homologue of the endoplasmic reticulum, interacts with its non-native substrate proteins in an ATP-dependent manner. This interaction is coupled to the ATPase cycle of the chaperone. Binding of short, synthetic peptides stimulate the ATPase activity of BiP. In previous work, we showed that a stably unfolded antibody domain forms a binary complex with BiP. In this study we made use of this complex to analyse the effect of substrate proteins on the ATPase cycle of BiP. Kinetic constants of the partial reactions of the ATPase cycle were determined without substrate, in the presence of a short binding peptide and in the presence of the antibody domain. We show that, in contrast to smaller peptides, the non-native protein domain decelerates the rate limiting hydrolysis step of the ATPase cycle.     

Analysis of molecular interactions in heat-induced aggregation of a non-inhibitory serpin ovalbumin using a molecular chaperone

Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a Km of 165 microM and a Vmax of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a Kd of 0.52 microM by BiP, lower than that for RCMLA (Kd = 1.1 microM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (Kd = 21 microM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.     

Isolation of proteins that speifically interact with the ATPase domain of mammalian ER chaperone,BiP

BiP, immunoglobulin binding protein, is an ER homologue of Hsp 70. However, unlike other Hsp70 proteins, regulatory protein(s) for BiP has not been identified. Here, we demonstrated the presence of potential regulatory proteins for BiP using a pull-down assay. Since BiP can bind any unfolded protein, only the ATPase domain of BiP was used for the pull-down assay in order to minimize nonspecific binding. The ATPase domain was cloned to produce recombinant protein, which was then conjugated to CNBr-activated agarose. The structural conformation and ATP hydrolysis activity of the recombinant ATPase domain were similar to those of the native protein. Eight proteins from metabolically labeled mouse plasmacytoma cells specifically bound to the recombinant ATPase protein. The binding of these proteins was inhibited by excess amounts of free ATPase protein, and was dependent on the presence of ATP. These proteins were eluted by ADP. Of these proteins, Grp 170 and BiP where identified, while the others were not identified as known ER proteins, from Western blot analyses. The presence of the ATPase-binding proteins for Bip was first demonstrated in this study, and our data suggest similar regulatory machinery for BiP may exist in the ER, as found in prokaryotes and other cellular compartments.     

The Large Hsp70 Grp170 Binds to Unfolded Protein Substrates in Vivo with a Regulation Distinct from Conventional Hsp70s

The Hsp70 superfamily is a ubiquitous chaperone class that includes conventional and large Hsp70s. BiP is the only conventional Hsp70 in the endoplasmic reticulum (ER) whose functions include: assisting protein folding, targeting misfolded proteins for degradation, and regulating the transducers of the unfolded protein response. The ER also possesses a single large Hsp70, the glucose-regulated protein of 170 kDa (Grp170). Like BiP it is an essential protein, but its cellular functions are not well understood. Here we show that Grp170 can bind directly to a variety of incompletely folded protein substrates in the ER, and as expected for a bona fide chaperone, it does not interact with folded secretory proteins. Our data demonstrate that Grp170 and BiP associate with similar molecular forms of two substrate proteins, but while BiP is released from unfolded substrates in the presence of ATP, Grp170 remains bound. In comparison to conventional Hsp70s, the large Hsp70s possess two unique structural features: an extended C-terminal α-helical domain and an unstructured loop in the putative substrate binding domain with an unknown function. We find that in the absence of the α-helical domain the interaction of Grp170 with substrates is reduced. In striking contrast, deletion of the unstructured loop results in increased binding to substrates, suggesting the presence of unique intramolecular mechanisms of control for the chaperone functions of large Hsp70s.     

Substrate binding to the molecular chaperone Hsp104 and its regulation by nucleotides

The Hsp104 protein from Saccharomyces cerevisiae is a member of the Hsp100/Clp family of molecular chaperones. It mediates the solubilization of aggregated proteins in an ATP-dependent process assisted by the Hsp70/40 system. Although the principal function of Hsp104 is well established, the mechanistic details of this catalyzed disaggregation are poorly understood. In this work, we have investigated the interaction of Hsp104 with reduced, carboxymethylated alpha-lactalbumin (RCMLa), a permanently unfolded model substrate. Our results demonstrate that the affinity of Hsp104 toward polypeptides is regulated by nucleotides. In the presence of ATP or adenosine-5' -O-(3-thiotriphosphate), the chaperone formed complexes with RCMLa, whereas no binding was observed in the presence of ADP. In particular, the occupation of the N-terminally located nucleotide-binding domain with ATP seems to be crucial for substrate interaction. When ATP binding to this domain was impaired by mutation, Hsp104 lost its ability to interact with RCMLa. Our results also indicate that upon association with a polypeptide, a conformational change occurs within Hsp104 that strongly reduces the dynamics of nucleotide exchange and commits the bound polypeptide to ATP hydrolysis.     

BiP-binding sequences in HIV gp160. Implications for the binding specificity of bip.

BiP, a resident endoplasmic reticulum member of the HSP70 family of molecular chaperones, associates transiently with a wide variety of newly synthesized exocytotic proteins. In addition to immunoglobulin heavy and light chains, the first natural substrates identified for BiP, a number of viral polypeptides including the human immunodeficiency virus type 1 envelope glycoprotein gp160 interact with BiP during their passage through the endoplasmic reticulum. We have used a computer algorithm developed to predict BiP-binding sites within protein primary sequences to identify sites within gp160 that might mediate its association with BiP. Analysis of the ability of 22 synthetic heptapeptides corresponding to predicted binding sites to stimulate the ATPase activity of BiP or to compete with an unfolded polypeptide for binding to BiP indicated that about half of them are indeed recognized by the chaperone. All of the confirmed binding sites are localized within conserved regions of gp160, suggesting a conserved role for BiP in the folding of gp160. Information on the characteristics of confirmed BiP-binding peptides gained in this and previous studies has been utilized to improve the predictive power of the BiP Score algorithm and to investigate the differences in peptide binding specificities of HSP70 family members.     

Regulated release of ERdj3 from unfolded proteins by BiP

DnaJ proteins often bind to unfolded substrates and recruit their Hsp70 partners. This induces a conformational change in the Hsp70 that stabilizes its binding to substrate. By some unknown mechanism, the DnaJ protein is released. We examined the requirements for the release of ERdj3, a mammalian ER DnaJ, from substrates and found that BiP promoted the release of ERdj3 only in the presence of ATP. Mutations in ERdj3 or BiP that disrupted their interaction interrupted the release of ERdj3. BiP mutants that were defective in any step of the ATPase cycle were also unable to release ERdj3. These results demonstrate that a functional interaction between ERdj3 and BiP, including both a direct interaction and the ability to stimulate BiP's ATPase activity are required to release ERdj3 from substrate and support a model where ERdj3 must recruit BiP and stimulate its high-affinity association with the substrate through activation of ATP hydrolysis to trigger its own release from substrates. On the basis of similarities among DnaJs and Hsp70s, this is likely to be applicable to other Hsp70-DnaJ pairs.     

Divergent functional properties of the ribosome-associated molecular chaperone Ssb compared with other Hsp70s.

Ssbs of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, which can be cross-linked to nascent polypeptide chains. Because Ssbs are members of a divergent subclass of Hsp70s found thus far only in fungi, we asked if the structural requirements for in vivo function were similar to those of "classic" Hsp70s. An intact peptide-binding domain is essential and an alteration of a conserved residue in the peptide-binding cleft (V442) affects function. However, Ssb tolerates a number of alterations in the peptide-binding cleft, revealing a high degree of flexibility in its functional requirements. Because binding of Ssb to peptide substrates in vitro was undetectable, we assessed the importance of substrate binding using the chimera BAB, in which the peptide binding domain of Ssb is exchanged for the analogous domain of the more "classical" Hsp70, Ssa. BAB, which binds peptide substrates in vitro, can substitute for Ssb in vivo. Alteration of a residue in the peptide-binding cleft of BAB creates a protein with a reduced affinity for peptide and altered ribosome binding that is unable to substitute for Ssb in vivo. These results indicate that Ssb's ability to bind unfolded polypeptides is likely critical for its function. This binding accounts, in part, for its stable interaction with translating ribosomes, even although it has a low affinity for peptides that detectably bind to other Hsp70s in vitro. These unusual properties may allow Ssb to function efficiently as a chaperone for ribosome-bound nascent chains.     

Neurospora crassa FKBP22 is a novel ER chaperone and functionally cooperates with BiP

FK506 binding proteins (FKBPs) belong to the family of peptidyl prolyl cis-trans isomerases (PPIases) catalyzing the cis/trans isomerisation of Xaa-Pro bonds in oligopeptides and proteins. FKBPs are involved in folding, assembly and trafficking of proteins. However, only limited knowledge is available about the roles of FKBPs in the endoplasmic reticulum (ER) and their interaction with other proteins. Here we show the ER located Neurospora crassa FKBP22 to be a dimeric protein with PPIase and a novel chaperone activity. While the homodimerization of FKBP22 is mediated by its carboxy-terminal domain, the amino-terminal domain is a functional FKBP domain. The chaperone activity is mediated by the FKBP domain but is exhibited only by the full-length protein. We further demonstrate a direct interaction between FKBP22 and BiP, the major Hsp70 chaperone in the ER. The binding to BiP is mediated by the FKBP domain of FKBP22. Interestingly BiP enhances the chaperone activity of FKBP22. Both proteins form a stable complex with an unfolded substrate protein and thereby prevent its aggregation. These results suggest that BiP and FKBP22 form a folding helper complex with a high chaperoning capacity in the ER of Neurospora crassa.     

Defining the structure of the substrate-free state of the DnaK molecular chaperone

Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain.     

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.     

The crystal structure of the human co-chaperone P58(IPK)

P58^IPK is one of the endoplasmic reticulum- (ER-) localised DnaJ (ERdj) proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58^IPK, expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58^IPK, and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58^IPK to 3.0 Å resolution using a combination of molecular replacement and single wavelength anomalous diffraction. The structure shows the human P58^IPK monomer to have a very elongated overall shape. In addition to the conserved J domain, P58^IPK contains nine N-terminal tetratricopeptide repeat motifs, divided into three subdomains of three motifs each. The J domain is attached to the C-terminal end via a flexible linker, and the structure shows the conserved Hsp70-binding histidine-proline-aspartate (HPD) motif to be situated on the very edge of the elongated protein, 100 Å from the putative binding site for unfolded protein substrates. The residues that comprise the surface surrounding the HPD motif are highly conserved in P58^IPK from other organisms but more varied between the human ERdj proteins, supporting the view that their regulation of different BiP functions is facilitated by differences in BiP-binding.     

BAG-1 modulates the chaperone activity of Hsp70/Hsc70.

The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG-1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy-terminal peptide-binding domain, and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG-1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG-1 inhibits Hsp/Hsc70-mediated in vitro refolding of an unfolded protein substrate, whereas BAG-1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG-1 to one of its known cellular targets, Bcl-2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG-1 also protected certain cell lines from heat shock-induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG-1 may explain the diverse interactions observed between BAG-1 and several other proteins, including Raf-1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG-1 on Hsp/Hsc70 chaperone activity suggest that BAG-1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.     

NMR investigations of allosteric processes in a two-domain Thermus thermophilus Hsp70 molecular chaperone

Hsp70 chaperones are two-domain proteins that assist in intra-cellular protein (re) folding processes in all species. The protein folding activity of the substrate binding domain of the Hsp70s is regulated by nucleotide binding at the nucleotide-binding domain through an as yet undefined heterotropic allosteric mechanism. The available structures of the isolated domains of Hsp70s have given very limited indications of nucleotide-induced conformational changes that could modulate the affinity for substrate proteins. Here, we present a multi-dimensional NMR study of a prokaryotic Hsp70 homolog, Thermus thermophilus DnaK, using a 54kDa construct containing both nucleotide binding domain and most of the substrate binding domain. It is determined that the nucleotide binding domain and substrate binding domain are closely associated in all ligand states studied. Comparison of the assigned NMR spectra of the two-domain construct with those of the previously studied isolated nucleotide binding domain, allowed the identification of the nucleotide binding domain-substrate binding domain interface. A global three-dimensional structure was obtained for the two-domain construct on the basis of this information and of NMR residual dipolar couplings measurements. This is the first experimental elucidation of the relative positioning of the nucleotide binding domain and substrate binding domain for any Hsp70 chaperone. Comparisons of NMR data between various ligand states including nucleotide-free, ATP, ADP.Pi and ADP.Pi+ peptide bound, identified residues involved in the allosteric inter-domain communication. In particular, peptide binding to the substrate binding domain was found to cause conformational changes in the NBD extending to the nucleotide binding pocket. Detailed analysis suggests that the inter-domain interface becomes tighter in the (nucleotide binding domain ligation/substrate binding domain ligation) order ATP/apo, ADP.Pi/apo ADP.Pi/peptide.     

BiPPred: Combined sequence‐ and structure‐based prediction of peptide binding to the Hsp70 chaperone BiP

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi‐scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence‐based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence‐based prediction models were fitted using this and other peptide binding data. A structure‐based position‐specific scoring matrix (SB‐PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB‐PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA‐based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi‐scale pipeline can readily be applied to other protein‐peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence‐based prediction models is not available. Proteins 2016; 84:1390–1407. © 2016 Wiley Periodicals, Inc.     

Interaction of BiP with the J-domain of the Sec63p component of the endoplasmic reticulum protein translocation complex.

Proteins of the Hsp70 family of ATPases interact with a conserved domain of their J-protein partners, the J-domain, to function in numerous cellular processes. We have studied the interaction of BiP, an Hsp70 family member in the lumen of the endoplasmic reticulum, with the J-domain of Sec63p, a component of the Sec complex involved in post-translational protein translocation across the endoplasmic reticulum membrane. In a real-time solid phase binding assay, BiP binds to the immobilized Sec complex or to a fusion protein of the J-domain and glutathione S-transferase in a reaction that requires ATP hydrolysis. In the final complex, BiP is bound in the ADP form with its peptide binding pocket occupied. An intact peptide binding pocket is required for this interaction. Our experiments suggest that the activation of BiP by the J-domain involves a transient contact between these components, and that in the absence of physiological substrates, J-activated BiP binds even to the J-proteins themselves.     

A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1

In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm. We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1. It is still unclear how this event contributes to the overall regulation of Ire1. The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity. The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain. Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins. Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature. We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses.     

In vivo threonine phosphorylation of immunoglobulin binding protein (BiP) maps to its protein binding domain

lmmunoglobin binding protein (BiP) molecules exist as both monomers and oligomers and phosphorylated BiP is restricted to the oligomeric pool. Modified BiP is not bound to proteins such as immunoglobulin heavy chain and consequently, may constitute an inactive form. Unlike earlier analysis of mammalian BiP isolated by two-dimensional gel electrophoresis, results here demonstrated that immunoprecipitated BiP displayed predominantly threonine phosphorylation with only a trace of detectable phosphoserine. Like other Hsp70 family members, BiP is comprised of three domains: an amino terminal domain which binds nucleotide, an 18 kilodalton domain which binds peptide, and a carboxyl terminal variable domain of unknown function. Cyanogen bromide cleavage and enzymatic digestion experiments mapped threonine phosphorylation to a site within a 47 amino acid sequence of the peptide binding domain which contains seven threonine residues. Partial proteinase K digestion in the presence of ATP independently verified that the in vivo phosphorylation site of mammalian (BiP) is located within the peptide binding domain. Furthermore, phosphorylation did not impede BiPs ATP-induced conformational change. Thus, the peptide binding domain of BiP is phosphorylated on threonine residue(s) mapping to not more than two tryptic fragments within the peptide binding domain. This location on the molecule could explain why phosphorylated BiP is not detected bound to proteins in vivo.     

An unstructured C-terminal region of the Hsp90 co-chaperone p23 is important for its chaperone function.

p23 is a co-chaperone of the heat shock protein Hsp90. p23 binds to Hsp90 in its ATP-bound state and, on its own, interacts specifically with non-native proteins. In our attempt to correlate these functions to specific regions of p23 we have identified an unstructured region in p23 that maps to the C-terminal part of the protein sequence. This unstructured region is dispensible for interaction of p23 with Hsp90, since truncated p23 can still form complexes with Hsp90. In contrast, however, truncation of the C-terminal 30 amino acid residues of p23 affects the ability of p23 to bind non-native proteins and to prevent their non-specific aggregation. The isolated C-terminal region itself is not able to act as a chaperone nor is it possible to complement truncated p23 by addition of this peptide. These results imply that the binding site for Hsp90 is contained in the folded domain of p23 and that for efficient interaction of p23 with non-native proteins both the folded domain and the C-terminal unstructured region are required.     

Localization of the chaperone domain of FKBP52

FKBP52, a multidomain peptidyl prolyl cis/trans-isomerase (PPIase), is found in complex with the chaperone Hsp90 and the co-chaperone p23. It displays both PPIase and chaperone activity in vitro. To localize these two activities to specific regions of the protein, we created and analyzed a set of fragments of FKBP52. The PPIase activity toward both peptides and proteins is confined entirely to domain 1 (amino acids 1-148). The chaperone activity, however, resides in the C-terminal part of FKBP52, mainly in the region between amino acids 264 and 400 (domain 3). Interestingly, this domain also contains the tetratricopeptide repeats, which are responsible for the binding to C-terminal amino acids of Hsp90. Competition assays with a C-terminal Hsp90 peptide suggest that the non-native protein and Hsp90 are bound by different regions within this domain.