Invariant Glutamine Research Articles

Cdc14 phosphatases use an intramolecular pseudosubstrate motif to stimulate and regulate catalysis

Cdc14 phosphatases are related structurally and mechanistically to protein tyrosine phosphatases (PTPs) but evolved a unique specificity for phosphoSer-Pro-X-Lys/Arg sites primarily deposited by cyclin-dependent kinases. This specialization is widely conserved in eukaryotes. The evolutionary reconfiguration of the Cdc14 active site to selectively accommodate phosphoSer-Pro likely required modification to the canonical PTP catalytic cycle. While studying Saccharomyces cerevisiae Cdc14, we discovered a short sequence in the disordered C terminus, distal to the catalytic domain, which mimics an optimal substrate. Kinetic analyses demonstrated this pseudosubstrate binds the active site and strongly stimulates rate-limiting phosphoenzyme hydrolysis, and we named it "substrate-like catalytic enhancer" (SLiCE). The SLiCE motif is found in all Dikarya fungal Cdc14 orthologs and contains an invariant glutamine, which we propose is positioned via substrate-like contacts to assist orientation of the hydrolytic water, similar to a conserved active site glutamine in other PTPs that Cdc14 lacks. AlphaFold2 predictions revealed vertebrate Cdc14 orthologs contain a conserved C-terminal alpha helix bound to the active site. Although apparently unrelated to the fungal sequence, this motif also makes substrate-like contacts and has an invariant glutamine in the catalytic pocket. Altering these residues in human Cdc14A and Cdc14B demonstrated that it functions by the same mechanism as the fungal motif. However, the fungal and vertebrate SLiCE motifs were not functionally interchangeable, illuminating potential active site differences during catalysis. Finally, we show that the fungal SLiCE motif is a target for phosphoregulation of Cdc14 activity. Our study uncovered evolution of an unusual stimulatory pseudosubstrate motif in Cdc14 phosphatases.

The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of nine amino acids (GFXXPXPIQ) with an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

The Q Motif is Involved in DNA Binding in ChlR1 (DDX11) Helicase

Helicases are molecular motors that couple the energy of nucleoside triphosphate (NTP) hydrolysis to the unwinding and remodeling of structured DNA or RNA. The conversion of energy derived from NTP hydrolysis into unwinding of double‐stranded nucleic acids is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Previous studies have suggested that the Q motif is involved in ATP binding. Mutations in the human ChlR1 (DDX11) gene are associated with Warsaw Breakage Syndrome characterized by cellular defects in genome maintenance. ChlR1 is known to play essential roles to preserve genomic stability, particularly in sister chromatid cohesion; however, its molecular mechanism remains unknown. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. The ChlR1‐Q23A mutant abolished helicase activity of ChlR1, and further DNA binding assays suggested the Q motif affected the DNA binding ability of ChlR1. ATP hydrolysis assays revealed that the ChlR1‐Q23A had impaired ATP hydrolysis activity. However, further ATP binding assays showed that the Q motif is not essential for ATP binding. The Q motif in FANCJ helicase, a ChlR1 sequence homology protein, regulates FANCJ's dimerization, while our size exclusion chromatography indicated that ChlR1 protein functions as monomer. Taken together, our results suggest that the Q motif in ChlR1 helicase is involved in DNA binding but not ATP binding.This work was supported in part by the Natural Sciences and Engineering Research Council of Canada (YW)

An update view on the substrate recognition mechanism of phosphodiesterases: A computational study of PDE10 and PDE4 bound with cyclic nucleotides

Early studies strongly implied that the specificity of cyclic nucleotide phosphodiesterases (PDEs) toward its endogenous substrates can be uniquely determined by the amido orientation of the invariant glutamine locating in the binding pocket of the enzyme. However, recently solved crystal structures of PDE4 (cAMP specific) and PDE10 (dual specific) in the presence of endogenous substrates have revealed that their invariant glutamine orientations are very similar despite exhibiting different substrate specificities proven physiologically. To understand this subtle specificity issue in the PDE family, here several experimentally inaccessible PDE-substrate complex models have been studied computationally, and the results are juxtaposed and compared in detail. Modeling results show that PDE10 in fact favors cAMP energetically but still can bind to cGMP owing to the robust hydrogen-bond network in the vicinity of the invariant glutamine side chain. PDE4 fails to accommodate cGMP is correlated to the weakening of this same hydrogen-bond network but not owing to any steric strain in the binding pocket. An Asn residue in the binding pocket of PDE4 has enhanced the specificity of the binding to cAMP sideway as observed in our computer simulation. Further to the previously studied syn- versus anti-conformational specificity of cAMP in PDE10, the unexpected substrate-binding mode in PDE10 versus PDE4 as reported here strongly suggested that there are remaining uncertainties in the substrate orientation and recognition mechanism in the PDE families. The molecular details of the binding pocket observed in this study provide hints for more optimal PDE4 and PDE10 inhibitor design.

The Q Motif of Fanconi Anemia Group J Protein (FANCJ) DNA Helicase Regulates Its Dimerization, DNA Binding, and DNA Repair Function

The Q motif, conserved in a number of RNA and DNA helicases, is proposed to be important for ATP binding based on structural data, but its precise biochemical functions are less certain. FANCJ encodes a Q motif DEAH box DNA helicase implicated in Fanconi anemia and breast cancer. A Q25A mutation of the invariant glutamine in the Q motif abolished its ability to complement cisplatin or telomestatin sensitivity of a fancj null cell line and exerted a dominant negative effect. Biochemical characterization of the purified recombinant FANCJ-Q25A protein showed that the mutation disabled FANCJ helicase activity and the ability to disrupt protein-DNA interactions. FANCJ-Q25A showed impaired DNA binding and ATPase activity but displayed ATP binding and temperature-induced unfolding transition similar to FANCJ-WT. Size exclusion chromatography and sedimentation velocity analyses revealed that FANCJ-WT existed as molecular weight species corresponding to a monomer and a dimer, and the dimeric form displayed a higher specific activity for ATPase and helicase, as well as greater DNA binding. In contrast, FANCJ-Q25A existed only as a monomer, devoid of helicase activity. Thus, the Q motif is essential for FANCJ enzymatic activity in vitro and DNA repair function in vivo.

Structural asymmetry of phosphodiesterase-9, potential protonation of a glutamic acid, and role of the invariant glutamine.

PDE9 inhibitors show potential for treatment of diseases such as diabetes. To help with discovery of PDE9 inhibitors, we performed mutagenesis, kinetic, crystallographic, and molecular dynamics analyses on the active site residues of Gln453 and its stabilizing partner Glu406. The crystal structures of the PDE9 Q453E mutant (PDE9Q453E) in complex with inhibitors IBMX and (S)-BAY73-6691 showed asymmetric binding of the inhibitors in two subunits of the PDE9Q453E dimer and also the significant positional change of the M-loop at the active site. The kinetic analysis of the Q453E and E406A mutants suggested that the invariant glutamine is critical for binding of substrates and inhibitors, but is unlikely to play a key role in the differentiation between substrates of cGMP and cAMP. The molecular dynamics simulations suggest that residue Glu406 may be protonated and may thus explain the hydrogen bond distance between two side chain oxygens of Glu453 and Glu406 in the crystal structure of the PDE9Q453E mutant. The information from these studies may be useful for design of PDE9 inhibitors.

Structural Asymmetry of Phosphodiesterase-9, Potential Protonation of a Glutamic Acid, and Role of the Invariant Glutamine

Structural Asymmetry of Phosphodiesterase-9, Potential Protonation of a Glutamic Acid, and Role of the Invariant Glutamine

Interactions between Cyclic Nucleotide Phosphodiesterase 11 Catalytic Site and Substrates or Tadalafil and Role of a Critical Gln-869 Hydrogen Bond

Poor understanding of the topography of cyclic nucleotide (CN) phosphodiesterase (PDE) catalytic sites compromises development of potent, selective inhibitors for therapeutic use. In the X-ray crystal structures of the catalytic domains of some PDEs, an invariant glutamine hydrogen bonds with groups at C6 and N1 or N7 on catalytic products or analogous positions of some inhibitors, inferring similar bonds with CNs (Nature 425:98-102, 2003; J Mol Biol 337:355-365, 2004; Mol Cell 15:279-286, 2004). A site-directed mutant (Q869A) lacking this invariant Gln in cGMP-/cAMP-hydrolyzing PDE11 had unaltered catalytic activity and affinity for sildenafil; but cGMP/cAMP or tadalafil affinity was reduced approximately 50- or 140-fold, respectively, and calculated free energy of binding suggested one hydrogen bond for each. A cGMP analog lacking the C6 oxygen had approximately 80-fold weakened affinity, modifications at N(2), N7, or 2'-OH diminished affinity approximately 16-fold, and analogs with groups appended at N1 had only 2- to 6-fold weakened affinity. Analogs with C8 substitutions were ineffective inhibitors, suggesting that cGMP binds in the anti conformation. Calculated decline in free energy of binding was consistent with that for one hydrogen bond only in the analog lacking binding potential at C6. In conclusion, Gln-869 interacts strongly with cGMP/cAMP and tadalafil, but not with sildenafil; interactions with CN analogs suggest a hydrogen bond only between Gln-869 and the C6 substituent. The results define interactions between the PDE11 catalytic site and substrates/inhibitors and advance potential for inhibitor design.

An insight into the pharmacophores of phosphodiesterase-5 inhibitors from synthetic and crystal structural studies

Selective inhibitors of cyclic nucleotide phosphodiesterase-5 (PDE5) have been used as drugs for treatment of male erectile dysfunction and pulmonary hypertension. An insight into the pharmacophores of PDE5 inhibitors is essential for development of second generation of PDE5 inhibitors, but has not been completely illustrated. Here we report the synthesis of a new class of the sildenafil derivatives and a crystal structure of the PDE5 catalytic domain in complex with 5-(2-ethoxy-5-(sulfamoyl)-3-thienyl)-1-methyl-3-propyl-1,6-dihydro-7 H-pyrazolo[4,3- d]pyrimidin-7-one (1 2). Inhibitor 12 induces conformational change of the H-loop (residues 660–683), which is different from any of the known PDE5 structures. The pyrazolopyrimidinone groups of 12 and sildenafil are well superimposed, but their sulfonamide groups show a positional difference of as much as 1.5 Å. The structure–activity analysis suggests that a small hydrophobic pocket and the H-loop of PDE5 are important for the inhibitor affinity, in addition to two common elements for binding of almost all the PDE inhibitors: the stack against the phenylalanine and the hydrogen bond with the invariant glutamine. However, the PDE5– 12 structure does not provide a full explanation to affinity changes of the inhibitors. Thus alternatives such as conformational change of the M-loop are open and further structural study is required.

Structures of the four subfamilies of phosphodiesterase-4 provide insight into the selectivity of their inhibitors

PDE4 (phosphodiesterase-4)-selective inhibitors have attracted much attention as potential therapeutics for the treatment of both depression and major inflammatory diseases, but their practical application has been compromised by side effects. A possible cause for the side effects is that current PDE4-selective inhibitors similarly inhibit isoforms from all four PDE4 subfamilies. The development of PDE4 subfamily-selective inhibitors has been hampered by a lack of structural information. In the present study, we rectify this by providing the crystal structures of the catalytic domains of PDE4A, PDE4B and PDE4D in complex with the PDE4 inhibitor NVP {4-[8-(3-nitrophenyl)-[1,7]naphthyridin-6-yl]benzoic acid} as well as the unliganded PDE4C structure. NVP binds in the same conformation to the deep cAMP substrate pocket and interacts with the same residues in each instance. However, detailed structural comparison reveals significant conformational differences. Although the active sites of PDE4B and PDE4D are mostly comparable, PDE4A shows significant displacements of the residues next to the invariant glutamine residue that is critical for substrate and inhibitor binding. PDE4C appears to be more distal from other PDE4 subfamilies, with certain key residues being disordered. Our analyses provide the first structural basis for the development of PDE4 subfamily-selective inhibitors.

Crystal structure of the Leishmania major phosphodiesterase LmjPDEB1 and insight into the design of the parasite-selective inhibitors.

Human leishmaniasis is a major public health problem in many countries, but chemotherapy is in an unsatisfactory state. Leishmania major phosphodiesterases (LmjPDEs) have been shown to play important roles in cell proliferation and apoptosis of the parasite. Thus LmjPDE inhibitors may potentially represent a novel class of drugs for the treatment of leishmaniasis. Reported here are the kinetic characterization of the LmjPDEB1 catalytic domain and its crystal structure as a complex with 3-isobutyl-1-methylxanthine (IBMX) at 1.55 A resolution. The structure of LmjPDEB1 is similar to that of human PDEs. IBMX stacks against the conserved phenylalanine and forms a hydrogen bond with the invariant glutamine, in a pattern common to most inhibitors bound to human PDEs. However, an extensive structural comparison reveals subtle, but significant differences between the active sites of LmjPDEB1 and human PDEs. In addition, a pocket next to the inhibitor binding site is found to be unique to LmjPDEB1. This pocket is isolated by two gating residues in human PDE families, but constitutes a natural expansion of the inhibitor binding pocket in LmjPDEB1. The structure particularity might be useful for the development of parasite-selective inhibitors for the treatment of leishmaniasis.

The Molecular Basis for Different Recognition of Substrates by Phosphodiesterase Families 4 and 10

Phosphodiesterases (PDEs) are key enzymes that control the cellular concentrations of the second messengers cAMP and cGMP. The mechanism for selective recognition of substrates cAMP and cGMP by individual PDE families remains a puzzle. To understand the mechanism for substrate recognition by PDE enzymes, the crystal structure of the catalytic domain of an inactive D201N mutant of PDE4D2 in complex with substrate cAMP has been determined at 1.56 Å resolution. The structure shows that Gln369 forms only one hydrogen bond with the adenine of cAMP. This finding provides experimental evidence against the hypothesis of two hydrogen bonds between the invariant glutamine and the substrate cAMP in PDE4, and thus suggests that the widely circulated “glutamine switch” model is unlikely the mechanism for substrate recognition by PDEs. A structure comparison between PDE4D2–cAMP and PDE10A2–cAMP reveals an anti configuration of cAMP in PDE4D2 but syn in PDE10A2, in addition to different contact patterns of cAMP in these two structures. These observations imply that individual PDE families have their characteristic mechanisms for substrate recognition.

Structural insight into substrate specificity of phosphodiesterase 10.

Phosphodiesterases (PDEs) hydrolyze the second messengers cAMP and cGMP. It remains unknown how individual PDE families selectively recognize cAMP and cGMP. This work reports structural studies on substrate specificity. The crystal structures of the catalytic domains of the D674A and D564N mutants of PDE10A2 in complex with cAMP and cGMP reveal that two substrates bind to the active site with the same syn configuration but different orientations and interactions. The products AMP and GMP bind PDE10A2 with the anti configuration and interact with both divalent metals, in contrast to no direct contact of the substrates. The structures suggest that the syn configurations of cAMP and cGMP are the genuine substrates for PDE10 and the specificity is achieved through the different interactions and conformations of the substrates. The PDE10A2 structures also show that the conformation of the invariant glutamine is locked by two hydrogen bonds and is unlikely to switch for substrate recognition. Sequence alignment shows a potential pocket, in which variation of amino acids across PDE families defines the size and shape of the pocket and thus determines the substrate specificity.

Phosphodiesterase 4 Inhibitors for the Treatment of Asthma and COPD

Type 4 cyclic nucleotide phosphodiesterases (PDE4s) are metallo-hydrolases which specifically hydrolyze cAMP to AMP in various cells types. The catalytic core is a bimetallic ion center composed of a tightly bound Zn(2+) and a loosely bound Mg(2+), which plays a dictating role in eliciting cAMP binding and catalysis activation. An invariant glutamine positioned opposite to the ion center serves as the substrate recognition determinant and synergizes the transient Mg-oxo-phosphate interaction in the substrate complex. The Mg(2+) binding is activated by a PKA-mediated serine phosphorylation and modulated through protein-protein interactions, thus, providing efficient mechanisms in the temporal regulation of cAMP signaling. Several PDE4 inhibitors including roflumilast, cilomilast and rolipram also rely on the interaction with the glutamine and metallic ion center for binding, with their affinity enhanced dramatically by the presence of the Mg(2+) ion. Recent studies have provided new insights into the role of this enzyme in inflammatory settings, CFTR regulation, long term potentiation, and its importance in immune surveillance. The major inflammatory cytokines which are modulated with PDE4 inhibitors include TNFalpha, IL-2, IFNgamma, IL-12, GM-CSF and LTB(4). The role of PDE4 inhibitors in modulating cytokines, lipid mediators and in mucociliary clearance, along with clinical efficacy in asthma and/or COPD demonstrated with roflumilast and cilomilast, suggest a broad anti-inflammatory spectrum for these compounds. Presently, the major impediment to approval of these novel therapies has been the mechanism based gastrointestinal adverse events which has limited the dosing and the ultimate efficacy with these novel therapeutic agents.

Enantiomer Discrimination Illustrated by the High Resolution Crystal Structures of Type 4 Phosphodiesterase

Type 4 phosphodiesterase (PDE4) inhibitors are emerging as new treatments for a number of disorders including asthma and chronic obstructive pulmonary disease. Here we report the biochemical characterization on the second generation inhibitor (+)-1 (L-, IC50=0.4 nM) and its enantiomer (-)-1 (L-, IC50=43 nM) and their cocrystal structures with PDE4D at 2.0 A resolution. Despite the 107-fold affinity difference, both enantiomers interact with the same sets of residues in the rigid active site. The weaker (-)-1 adopts an unfavorable conformation to preserve the pivotal interactions between the Mg-bound waters and the N-oxide of pyridine. These structures support a model in which inhibitors are anchored by the invariant glutamine at one end and the metal-pocket residues at another end. This model provides explanations for most of the observed structure-activity relationship and the metal ion dependency of the catechol-ether based inhibitors and should facilitate their further design.

Multiple Elements Jointly Determine Inhibitor Selectivity of Cyclic Nucleotide Phosphodiesterases 4 and 7

Phosphodiesterase (PDE) inhibitors have been widely studied as therapeutics for treatment of human diseases. However, the mechanism by which each PDE family recognizes selectively a category of inhibitors remains a puzzle. Here we report the crystal structure of PDE7A1 catalytic domain in complex with non-selective inhibitor 3-isobutyl-1-methylxanthine and kinetic analysis on the mutants of PDE7A1 and PDE4D2. Our studies suggest at least three elements play critical roles in inhibitor selectivity: 1) the conformation and position of an invariant glutamine, 2) the natures of scaffolding residues, and 3) residues that alter shape and size of the binding pocket. Kinetic analysis shows that single PDE7 to PDE4 mutations increase the sensitivity of PDE7 to PDE4 inhibitors but are not sufficient to render the engineered enzymes comparable with the wild types. The triple S373Y/S377T/I412S mutation of PDE7A1 produces a PDE4-like enzyme, implying that multiple elements must work together to determine inhibitor selectivity.

Structural Basis for the Activity of Drugs that Inhibit Phosphodiesterases

Phosphodiesterases (PDEs) comprise a large family of enzymes that catalyze the hydrolysis of cAMP or cGMP and are implicated in various diseases. We describe the high-resolution crystal structures of the catalytic domains of PDE4B, PDE4D, and PDE5A with ten different inhibitors, including the drug candidates cilomilast and roflumilast, for respiratory diseases. These cocrystal structures reveal a common scheme of inhibitor binding to the PDEs: (i) a hydrophobic clamp formed by highly conserved hydrophobic residues that sandwich the inhibitor in the active site; (ii) hydrogen bonding to an invariant glutamine that controls the orientation of inhibitor binding. A scaffold can be readily identified for any given inhibitor based on the formation of these two types of conserved interactions. These structural insights will enable the design of isoform-selective inhibitors with improved binding affinity and should facilitate the discovery of more potent and selective PDE inhibitors for the treatment of a variety of diseases.

A Glutamine Switch Mechanism for Nucleotide Selectivity by Phosphodiesterases

Phosphodiesterases (PDEs) comprise a family of enzymes that modulate the immune response, inflammation, and memory, among many other functions. There are three types of PDEs: cAMP-specific, cGMP-specific, and dual-specific. Here we describe the mechanism of nucleotide selectivity on the basis of high-resolution co-crystal structures of the cAMP-specific PDE4B and PDE4D with AMP, the cGMP-specific PDE5A with GMP, and the apo-structure of the dual-specific PDE1B. These structures show that an invariant glutamine functions as the key specificity determinant by a “glutamine switch” mechanism for recognizing the purine moiety in cAMP or cGMP. The surrounding residues anchor the glutamine residue in different orientations for cAMP and for cGMP. The PDE1B structure shows that in dual-specific PDEs a key histidine residue may enable the invariant glutamine to toggle between cAMP and cGMP. The structural understanding of nucleotide binding enables the design of new PDE inhibitors that may treat diseases in which cyclic nucleotides play a critical role.

The Q Motif: A Newly Identified Motif in DEAD Box Helicases May Regulate ATP Binding and Hydrolysis

SF1 and SF2 helicases have structurally conserved cores containing seven to eight distinctive motifs and variable amino- and carboxyl-terminal flanking sequences. We have discovered a motif upstream of motif I that is unique to and characteristic of the DEAD box family of RNA helicases. It consists of a 9 amino acid sequence containing an invariant glutamine. A conserved phenylalanine occurs 17 aa further upstream. Sequence alignments, site-specific mutagenesis, and ATPase assays show that this motif and the upstream phenylalanine are highly conserved, that they are essential for viability in the yeast Saccharomyces cerevisiae, and that they control ATP binding and hydrolysis in the yeast translation-initiation factor eIF4A. These results are consistent with computer studies of the solved crystal structures.

The Crystal Structure of the MJ0796 ATP-binding Cassette: IMPLICATIONS FOR THE STRUCTURAL CONSEQUENCES OF ATP HYDROLYSIS IN THE ACTIVE SITE OF AN ABC TRANSPORTER

The crystal structure of the MJ0796 ATP-binding cassette, a member of the o228/LolD transporter family, has been determined at 2.7-A resolution with MgADP bound at its active site. Comparing this structure with that of the ATP-bound form of the HisP ATP-binding cassette (Hung, L. W., Wang, I. X., Nikaido, K., Liu, P. Q., Ames, G. F., and Kim, S. H. (1998) Nature 396, 703-707) shows a 5-A withdrawal of a phylogenetically invariant glutamine residue from contact with the gamma-phosphate of ATP in the active site. This glutamine is located in a protein segment that links the rigid F(1)-type ATP-binding core of the enzyme to an ABC transporter-specific alpha-helical subdomain that moves substantially away from the active site in the MgADP-bound structure of MJ0796 compared with the ATP-bound structure of HisP. A similar conformational effect is observed in the MgADP-bound structure of MJ1267 (Karpowich, N., et al. (2001) Structure, in press), establishing the withdrawal of the glutamine and the coupled outward rotation of the alpha-helical subdomain as consistent consequences of gamma-phosphate release from the active site of the transporter. Considering this subdomain movement in the context of a leading model for the physiological dimer of cassettes present in ABC transporters indicates that it produces a modest mechanical change that is likely to play a role in facilitating nucleotide exchange out of the ATPase active site. Finally, it is noteworthy that one of the intersubunit packing interactions in the MJ0796 crystal involves antiparallel beta-type hydrogen bonding interactions between the outermost beta-strands in the two core beta-sheets, leading to their fusion into a single extended beta-sheet, a type of structural interaction that has been proposed to play a role in mediating the aggregation of beta-sheet-containing proteins.