The positive allosteric modulator BPAM344 and L-glutamate introduce an active-like structure of the ligand-binding domain of GluK2.

Kainate receptors belong to the family of ionotropic glutamate receptors and contribute to the majority of fast excitatory neurotransmission. Consequently, they also play a role in brain diseases. Therefore, understanding how these receptors can be modulated is of importance. Our study provides a crystal structure of the dimeric ligand-binding domain of the kainate receptor GluK2 in complex with L-glutamate and the small-molecule positive allosteric modulator, BPAM344, in an active-like conformation. The role of Thr535 and Gln786 in modulating GluK2 by BPAM344 was investigated using a calcium-sensitive fluorescence-based assay on transiently transfected cells expressing GluK2 and mutants hereof. This study may aid in the design of compounds targeting kainate receptors, expanding their potential as targets for the treatment of brain diseases.

The World of GPCR dimers - Mapping dopamine receptor D2 homodimers in different activation states and configuration arrangements.

G protein-coupled receptors (GPCRs) are known to dimerize, but the molecular and structural basis of GPCR dimers is not well understood. In this study, we developed a computational framework to generate models of symmetric and asymmetric GPCR dimers using different monomer activation states and identified their most likely interfaces with molecular details. We chose the dopamine receptor D2 (D2R) homodimer as a case study because of its biological relevance and the availability of structural information. Our results showed that transmembrane domains 4 and 5 (TM4 and TM5) are mostly found at the dimer interface of the D2R dimer and that these interfaces have a subset of key residues that are mostly nonpolar from TM4 and TM5, which was in line with experimental studies. In addition, TM2 and TM3 appear to be relevant for D2R dimers. In some cases, the inactive configuration is unaffected by the partnered protomer, whereas in others, the active protomer adopts the properties of an inactive receptor. Additionally, the ß-arrestin configuration displayed the properties of an active receptor in the absence of an agonist, suggesting that a switch to another meta-state during dimerization occurred. Our findings are consistent with the experimental data, and this method can be adapted to study heterodimers and potentially extended to include additional proteins such as G proteins or ß-arrestins. In summary, this approach provides insight into the impact of the conformational status of partnered protomers on the overall quaternary GPCR macromolecular structure and dynamics.

C-Terminal ß8-α9 Interaction Modulates Thermal Stability and Enzymatic Activity Differently in Hyperthermophilic Esterase EstE1 and Mesophilic Esterase rPPE.

Hydrophobic interactions and hydrogen bonds are 2 types of noncovalent interactions that play distinct roles in the folding and structural stability of proteins. However, the specific roles of these interactions in hydrophobic or hydrophilic environments in α/ß-hydrolases are not fully understood. A hyperthermophilic esterase EstE1 in a dimer maintains the C-terminal ß8-α9 strand-helix via hydrophobic interactions (Phe276 and Leu299), constituting a closed dimer interface. Moreover, a mesophilic esterase rPPE in a monomer maintains the same strand-helix via a hydrogen bond (Tyr281 and Gln306). Unpaired polar residues (F276Y in EstE1 and Y281A/F and Q306A in rPPE) or reduced hydrophobic interactions (F276A/L299A in EstE1) between the ß8-α9 strand-helix decrease thermal stability. EstE1 (F276Y/L299Q) and rPPE WT, both with the ß8-α9 hydrogen bond, showed the same thermal stability as EstE1 WT and rPPE (Y281F/Q306L), which possess hydrophobic interactions instead. However, EstE1 (F276Y/L299Q) and rPPE WT exhibited higher enzymatic activity than EstE1 WT and rPPE (Y281F/Q306L), respectively. This suggests that α/ß-hydrolases favor the ß8-α9 hydrogen bond for catalytic activity in monomers or oligomers. Overall, these findings demonstrate how α/ß-hydrolases modulate hydrophobic interactions and hydrogen bonds to adapt to different environments. Both types of interactions contribute equally to thermal stability, but the hydrogen bond is preferred for catalytic activity. IMPORTANCE Esterases hydrolyze short to medium-chain monoesters and contain a catalytic His on a loop between the C-terminal ß8-strand and α9-helix. This study explores how hyperthermophilic esterase EstE1 and mesophilic esterase rPPE adapt to different temperatures by utilizing the ß8-α9 hydrogen bonds or hydrophobic interactions differently. EstE1 forms a hydrophobic dimer interface, while rPPE forms a monomer stabilized by a hydrogen bond. The study demonstrates that these enzymes stabilize ß8-α9 strand-helix differently but achieve similar thermal stability. While the ß8-α9 hydrogen bond or hydrophobic interactions contribute equally to thermal stability, the hydrogen bond provides higher activity due to increased catalytic His loop flexibility in both EstE1 and rPPE. These findings reveal how enzymes adapt to extreme environments while maintaining their functions and have implications for engineering enzymes with desired activities and stabilities.

Computational insight into in silico analysis and molecular dynamics simulation of the dimer interface residues of ALS-linked hSOD1 forms in apo/holo states: a combined experimental and bioinformatic perspective.

The aggregation of misfolded SOD1 proteins in neurodegenerative illnesses is a key pathological hallmark in amyotrophic lateral sclerosis (ALS). SOD1 is stabilized and enzymatically activated after binding to Cu/Zn and forming intramolecular disulfide. SOD1 aggregation/oligomerization is triggered by the dissociation of Cu and/or Zn ions. Therefore, we compared the possible effects of ALS-associated point mutations of the holo/apo forms of WT/I149T/V148G SOD1 variants located at the dimer interface to determine structural characterization using spectroscopic methods, computational approaches as well as molecular dynamics (MD) simulations. Predictive results of computational analysis of single-nucleotide polymorphisms (SNPs) suggested that mutant SOD1 has a deleterious effect on activity and structure destabilization. MD data analysis indicated that changes in flexibility, stability, hydrophobicity of the protein as well as increased intramolecular interactions of apo-SOD1 were more than holo-SOD1. Furthermore, a decrease in enzymatic activity in apo-SOD1 was observed compared to holo-SOD1. Comparative intrinsic and ANS fluorescence results of holo/apo-WT-hSOD1 and mutants indicated structural alterations in the local environment of tryptophan residue and hydrophobic patches, respectively. Experimental and MD data supported that substitution effect and metal deficiency of mutants (apo forms) in the dimer interface may promote the tendency to protein mis-folding and aggregation, consequently disrupting the dimer-monomer equilibrium and increased propensity to dissociation dimer into SOD-monomer ultimately leading to loss of stability and function. Overall, data analysis of apo/holo SOD1 forms on protein structure and function using computational and experimental studies will contribute to a better understanding of ALS pathogenicity.

Misfolding-Associated Exposure of Natively Buried Residues in Mutant SOD1 Facilitates Binding to TRAF6.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease primarily impacting motor neurons. Mutations in superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS. Several of these mutations lead to misfolding or toxic gain of function in the SOD1 protein. Recently, we reported that misfolded SOD1 interacts with TNF receptor-associated factor 6 (TRAF6) in the SOD1G93A rat model of ALS. Further, we showed in cultured cells that several mutant SOD1 proteins, but not wildtype SOD1 protein, interact with TRAF6 via the MATH domain. Here, we sought to uncover the structural details of this interaction through molecular dynamics (MD) simulations of a dimeric model system, coarse grained using the AWSEM force field. We used direct MD simulations to identify buried residues, and predict binding poses by clustering frames from the trajectories. Metadynamics simulations were also used to deduce preferred binding regions on the protein surfaces from the potential of the mean force in orientation space. Well-folded SOD1 was found to bind TRAF6 via co-option of its native homodimer interface. However, if loops IV and VII of SOD1 were disordered, as typically occurs in the absence of stabilizing Zn2+ ion binding, these disordered loops now participated in novel interactions with TRAF6. On TRAF6, multiple interaction hot-spots were distributed around the equatorial region of the MATH domain beta barrel. Expression of TRAF6 variants with mutations in this region in cultured cells demonstrated that TRAF6T475 facilitates interaction with different SOD1 mutants. These findings contribute to our understanding of the disease mechanism and uncover potential targets for the development of therapeutics.

Structural characterization of a dimerization interface in the CD28 transmembrane domain.

CD28 has a crucial role in regulating immune responses by enhancing T cell activation and differentiation. Recent studies have shown that the transmembrane helix (TMH) of CD28 mediates receptor assembly and activity, but a structural characterization of TMH is still lacking. Here, we determined the dimeric helix-helix packing of CD28-TMH using nuclear magnetic resonance (NMR) technology. Unexpectedly, wild-type CD28-TMH alone forms stable tetramers in lipid bicelles instead of dimers. The NMR structure of the CD28-TMH C165F mutant reveals that a GxxxA motif, which is highly conserved in many dimeric assemblies, is located at the dimerization interface. Mutating G160 and A164 can disrupt the transmembrane helix assembly and reduces CD28 enhancement in cells. In contrast, a previously proposed YxxxxT motif does not affect the dimerization of full-length CD28, but it does affect CD28 activity. These results imply that the transmembrane domain of CD28 regulates the signaling transduction in a complicated manner.

Comparative structural analysis of the human and Schistosoma glutathione transferase dimer interface using selective binding of bromosulfophthalein.

The binding channel of Schistosoma glutathione transferase (SGST) has been identified to possess a non-substrate site implicated in enzyme inhibition. This binding channel is formed by the interface of the GST dimer. We produced a comparative characterization of the SGST dimer interface with respect to that of human GST (hGST) analogues using the selective binding of bromosulfophthalein (BSP). First, two SGST and three hGST structures were used as search queries to assemble a data set of 48 empirical GST structures. Sequence alignment to generate a universal residue indexing scheme was then performed, followed by local superposition of the dimer interface. Principal component analysis revealed appreciable variation of the dimer interface, suggesting the potential for selective inhibition of SGST. BSP was found to dock invariably in the dimer interface core pocket, placing it in proximity to the GST catalytic domains, through which it may exert its inhibitory behavior. Binding poses across the GST forms were distinguished with ligand interaction profiling, where SGST complexes showed stabilization of ligand aromatic- and sulfonate moieties, which altogether anchor the ligand and produce a tight association. In comparison, missing aromatic stabilization in the hGST complexes impart large bonding distances, causing mobile poses likely to dissociate. Altogether, this study illustrates the potential for selective inhibition of SGST, rationalizes the selective behavior of the BSP inhibitor, and produces a reliable metric for construction and validation of pharmacophore models of the SGST binding channel.

Identification of mutation resistance coldspots for targeting the SARS-CoV2 main protease.

Mutations in the novel coronavirus SARS-CoV2 are the major concern as they might lead to drug/vaccine resistance. In the host cell, the virus largely depends on the main protease (Mpro ) to regulate infection hence it is one of the most attractive targets for inhibitor design. However, >19,000 mutations in the Mpro have already been reported. The mutations encompassing 282 amino acid positions and these "hotspots" might change the Mpro structure, activity and potentially delay therapeutic strategies targeting Mpro . Thus, here we identified 24 mutational "coldspots" where mutations have not been observed. We compared the structure-function relationship of these coldspots with several SARS-CoV2 Mpro X-ray crystal structures. We found that three coldspot residues (Leu141, Phe185, and Gln192) help to form the active site, while seven (Gly2, Arg4, Tyr126, Lys137, Leu141, Leu286, and Leu287) contribute to dimer formation that is required for Mpro activity. The surface of the dimer interface is more resistant to mutations compared to the active site. Interestingly, most of the coldspots are found in three clusters and forms conserved patterns when compared with other coronaviruses. Importantly, several conserved coldspots are available on the surface of the active site and at the dimer interface for targeting. The identification and short list of these coldspots offers a new perspective to target the SARS-CoV2 Mpro while avoiding mutation-based drug resistance.

A bispecific nanobody targeting the dimerization interface of epidermal growth factor receptor: Evidence for tumor suppressive actions in vitro and in vivo.

Targeting the dimer interface for the epidermal growth factor receptor (EGFR) that is highly conserved in the structure and directly involved in dimerization may solve the resistance problem that plagues anti-EGFR therapy. Heavy chain single domain antibodies have promising prospects as therapeutic antibodies. A bispecific nanobody was constructed based on previously screened humanized nanobodies that target the ß-loop at the EGFR dimer interface, an anti-FcγRIIIa (CD16) of natural killer cells (NK) nanobodies and anti-human serum albumin (HSA) nanobodies. The target gene was effectively expressed and secreted while controlled by promoter GAP in Pichia pastoris X33, and the expressed product was purified with a cation exchange and nickel chelation chromatography. The bispecific nanobody specifically bound to the surfaces of EGFR-overexpressed human epidermal carcinoma A431 cells and effectively inhibited tumor cell growth both in vitro and in vivo. In the A431 cell nude mouse xenograft model, the growth inhibition effect from the bispecific nanobody was significantly increased with the assistance of peripheral blood mononuclear cells (PBMCs), which was consistent with the results obtained in vitro, suggesting that there was an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. In addition, the intraperitoneal administration of bispecific nanobodies effectively reached tumor tissues in the shoulder dorsal region, but in significantly less distributed quantities than EGFR Dimer Nb77. To conclude, a bispecific nanobody targeting the EGFR dimer interface with ADCC effect was successfully constructed.

Redox requirements for ubiquitin-like urmylation of Ahp1, a 2-Cys peroxiredoxin from yeast.

The yeast peroxiredoxin Ahp1, like related anti-oxidant enzymes in other species, undergoes urmylation, a lysine-directed conjugation to ubiquitin-like modifier Urm1. Ahp1 assembles into a homodimer that detoxifies peroxides via forming intersubunit disulfides between peroxidatic and resolving cysteines that are subsequently reduced by the thioredoxin system. Although urmylation coincides with oxidative stress, it is unclear how this modification happens on a molecular level and whether it affects peroxiredoxin activity. Here, we report that thioredoxin mutants decrease Ahp1 urmylation in yeast and each subunit of the oxidized Ahp1 dimer is modified by Urm1 suggesting coupling of urmylation to dimerization. Consistently, Ahp1 mutants unable to form dimers, fail to be urmylated as do mutants that lack the peroxidatic cysteine. Moreover, Ahp1 urmylation involves at least two lysine residues close to the catalytic cysteines and can be prevented in yeast cells exposed to high organic peroxide concentrations. Our results elucidate redox requirements and molecular determinants critical for Ahp1 urmylation, thus providing insights into a potential link between oxidant defense and Urm1 utilization in cells.

Probing the Druggablility on the Interface of the Protein-Protein Interaction and Its Allosteric Regulation Mechanism on the Drug Screening for the CXCR4 Homodimer.

Modulating protein-protein interactions (PPIs) with small drug-like molecules targeting it exhibits great promise in modern drug discovery. G protein-coupled receptors (GPCRs) are the largest family of targeted proteins and could form dimers in living biological cells through PPIs. However, compared to drug development of the orthosteric site, there has been lack of investigations on the druggability of the PPI interface for GPCRs and its functional implication on experiments. Thus, in order to address these issues, we constructed a novel computational strategy, which involved in molecular dynamics simulation, virtual screening and protein structure network (PSN), to study one representative GPCR homodimer (CXCR4). One druggable pocket was identified in the PPI interface and one small molecule targeting it was screened, which could strengthen PPI mainly through hydrophobic interaction between the benzene rings of the PPI molecule and TM4 of the receptor. The PSN results further reveals that the PPI molecule could increase the number of the allosteric regulation pathways between the druggable pocket of the dimer interface to the orthostatic site for the subunit A but only play minor role for the other subunit B, leading to the asymmetric change in the volume of the binding pockets for the two subunits (increase for the subunit A and minor change for the subunit B). Consequently, the screening performance of the subunit A to the antagonists is enhanced while the subunit B is unchanged nearly, implying that the PPI molecule may be beneficial to enhance the drug efficacies of the antagonists. In addition, one main regulation pathway with the highest frequency was identified for the subunit A, which consists of Trp1955.34-Tyr190ECL2-Val1965.35-Gln2005.39-Asp2626.58-Cys28N-term, revealing their importance in the allosteric regulation from the PPI molecule. The observations from the work could provide valuable information for the development of the PPI drug-like molecule for GPCRs.

Biochemical characterization and mutational analysis of alanine racemase from Clostridium perfringens.

Clostridium perfringens is a gram-positive, anaerobic, pathogenic bacterium that can cause a wide range of diseases in humans, poultry and agriculturally important livestock. A pyridoxal-5-phosphate-dependent alanine racemase with a function in the racemization of d- and l-alanine is an attractive drug target for C. perfringens and other pathogens due to its absence in animals and humans. In this study alanine racemase from C. perfringens (CPAlr) was successfully expressed and purified in Escherichia coli and biochemically characterized. The purified CPAlr protein was a dimeric PLP-dependent enzyme with high substrate specificity. The optimal racemization temperature and pH were 40°C and 8.0, respectively. The kinetic parameters Km and kcat of CPAlr, determined by HPLC at 40°C were 19.1 mM and 17.2 s-1 for l-alanine, and 10.5 mM and 8.7 s-1 for d-alanine, respectively. Gel filtration chromatographic analysis showed that the molecular weight of mutant Y359A was close to monomeric form, suggesting that the inner layer residue Tyr359 might play an essential role in dimer-formation. Furthermore, the mutation at residues Asp171 and Tyr359 resulted in a dramatic increase in Km value and/or decreased in kcat value, indicating that the middle and inner layer residues Asp171 and Tyr359 of CPAlr might have the key role in substrate binding, catalytic activity or oligomerization state through the hydrogen-bonding interaction with the pentagonal ring waters and/or PLP cofactor.

Preparation and characterization of humanized nanobodies targeting the dimer interface of epidermal growth factor receptor (EGFR).

Epidermal growth factor receptor (EGFR) is an effective target for the treatment of many epithelial cancers. However, EGFR inhibitors have low clinical response rates and are prone to drug resistance arising from mutations and heterodimerization of EGFR. Therefore, targeting the highly conserved dimer interface of EGFR may be an effective strategy for improving the clinical response of anti-EGFR therapies. Nanobodies have significant advantages over conventional antibodies in terms of size, solubility, stability and cost-effectiveness. To investigate the feasibility of nanobodies targeting the dimer interface of EGFR as novel anticancer drugs, four nanobodies were screened from a commercial humanized nanobody phage antibody library using the EGFR237-267 peptide from the ß-hairpin loop of the dimer interface of EGFR as the antigen. A nanobody with an isoelectric point (pI) of 8.6, named EGFR dimer Nb77, was selected for further analysis of anticancer activities. EGFR dimer Nb77 was expressed in Escherichia coli Shuffle T7-B as a soluble (His)6-tagged protein and purified by a CM Sepharose column and a nickel-nitrilotriacetic acid (Ni-NTA) column. Purified EGFR dimer Nb77 could specifically bind to the surface of EGFR-overexpressing A431 cells in a dose-dependent and ligand-dependent manner, and this nanobody could effectively inhibit the growth of the tumour cells, with an inhibition rate similar to that of the monoclonal antibody EGFR dimer 5G9, which also targets the dimer interface of EGFR. This work is the first to prove that nanobodies targeting the dimer interface of EGFR have promising prospects as anticancer agents.

Hepatitis B virus core protein dimer­dimer interface is critical for viral replication.

Hepatitis B virus (HBV) core protein (HBc) serves pivotal roles in the viral life cycle, particularly serving as the basic unit for capsid assembly, and is closely associated with HBV genome replication and progeny virion production. Previous studies have demonstrated that HBc has at least two functional interfaces; two HBc monomers form a homodimer via an intradimer interface, and then 90 or 120 homodimers form an icosahedral capsid via a dimer­dimer interface. In the present study, the role of the HBc dimer­dimer interface in HBV replication was investigated. A panel of residues located at the dimer­dimer interface were identified based on the crystal structure of HBc. Native gel electrophoresis and western blotting revealed that, despite mutations in the dimer­dimer interface, HBc formed a capsid­like structure, whereas mutations at amino acid residues 23­39 completely disrupted capsid assembly. Using denaturing gel electrophoresis, Southern and Northern blotting, and quantitative polymerase chain reaction, it was demonstrated that none of the mutations in the dimer­dimer interface supported pregenomic RNA encapsidation or DNA replication. In addition, these mutants interacted with the wild-type (WT) HBc monomer and inhibited WT genome replication and virion production in a dose­dependent manner. However, the quantity of covalently closed circular DNA in the nucleus was not affected. The present study highlighted the importance of the HBc dimer­dimer interface for normal capsid function and demonstrated that the HBc dimer­dimer interface may be a novel antiviral target.

Bacterial Superantigen Toxins, CD28, and Drug Development.

During severe bacterial infections, death and disease are often caused by an overly strong immune response of the human host. Acute toxic shock is induced by superantigen toxins, a diverse set of proteins secreted by Gram-positive staphylococcal and streptococcal bacterial strains that overstimulate the inflammatory response by orders of magnitude. The need to protect from superantigen toxins led to our discovery that in addition to the well-known MHC class II and T cell receptors, the principal costimulatory receptor, CD28, and its constitutively expressed coligand, B7-2 (CD86), previously thought to have only costimulatory function, are actually critical superantigen receptors. Binding of the superantigen into the homodimer interfaces of these costimulatory receptors greatly enhances B7-2/CD28 engagement, leading to excessive pro-inflammatory signaling. This finding led to the design of short receptor dimer interface mimetic peptides that block the binding of superantigen and thus protect from death. It then turned out that such a peptide will protect also from Gram-negative bacterial infection and from polymicrobial sepsis. One such CD28 mimetic peptide is advancing in a Phase 3 clinical trial to protect from lethal wound infections by flesh-eating bacteria. These host-oriented therapeutics target the human immune system itself, rendering pathogens less likely to become resistant.

Spectral and structural analysis of large Stokes shift fluorescent protein dKeima570.

The Keima family comprises large Stokes shifts fluorescent proteins, which are useful for dual-color fluorescence crosscorrelation spectroscopy and multicolor imaging. dKeima570 belongs to the Keima family. It has a unique chromophore sequence composed of CYG with an emission peak at 570 nm, but its molecular properties are unclear. We report the spectral analysis of dKeima570 and its crystal structure at 2.0 Å resolution. The dKeima570 chromophore is mainly in the protonation state in the entire pH range. The pH-induced non-fluorescence state was observed below pH 4.0. The crystal structure of the dKeima570 chromophore has a cis conformation at pH 6.5. The chromophore is surrounded by a unique hydrogen bonding network containing a water bridge between Glu212 and Arg194. The analysis of the dimeric interface of dKeima570 revealed the key residues that maintain the oligomerization of Keima family. Structural comparisons of dKeima570 and mKeima provided insights into the unique large Stokes shifts characteristics of the Keima family.

Mapping the Interface of a GPCR Dimer: A Structural Model of the A2A Adenosine and D2 Dopamine Receptor Heteromer.

The A2A adenosine (A2AR) and D2 dopamine (D2R) receptors form oligomers in the cell membrane and allosteric interactions across the A2AR-D2R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A2AR-D2R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A2AR-D2R heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on A2AR-D2R receptor-receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the A2AR blocked heterodimer interactions and disrupted the allosteric effect of A2AR activation on D2R agonist binding. Protein-protein docking was used to construct a model of the A2AR-D2R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A2AR-D2R interactions in BRET experiments and altered the allosteric modulation. The heterodimer model provided insights into the structural basis of allosteric modulation and the technique developed to characterize the A2AR-D2R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.

Structure of Asp-bound peptidase E from Salmonella enterica: Active site at dimer interface illuminates Asp recognition.

Peptidase-E, a nonclassical serine peptidase, is specific for dipeptides with an N-terminal aspartate. This stringent substrate specificity remains largely unexplained. We report an aspartate-bound structure of peptidase-E at 1.83 Å resolution. In contrast to previous reports, the enzyme forms a dimer, and the active site is located at the dimer interface, well shielded from the solvent. Our findings further suggest that the stringent aspartate specificity of the enzyme is due to electrostatics and molecular complementarity in the active site. The new structural information presented herein may provide insights into the role of functionally important residues in peptidase-E.

The biological assembly of OXA-48 reveals a dimer interface with high charge complementarity and very high affinity.

Many class D ß-lactamases form dimers in solution. The functional basis of the dimerization of OXA-48-like class D ß-lactamases is not known, but in order to understand the structural requirements for dimerization of OXA-48, we have characterized the dimer interface. Size exclusion chromatography, small angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) were used to confirm the oligomeric state of OXA-48 in solution. X-ray crystallographic structures were used to elucidate the key interactions of dimerization. In silico residue scanning combined with site-directed mutagenesis was used to probe hot spots of dimerization. The affinity of dimerization was quantified using microscale thermophoresis, and the overall thermostability was investigated using differential scanning calorimetry. OXA-48 was consistently found to be a dimer in solution regardless of the method used, and the biological assembly found from the SAXS envelope is consistent with the dimer identified from the crystal structures. The buried chloride that interacts with Arg206 and Arg206' at the dimer interface was found to enhance the thermal stability by > 4 °C and crystal structures and mutations (R189A, R189A/R206A) identified several additional important ionic interactions. The affinity for OXA-48 R206A dimerization was in the picomolar range, thus revealing very high dimer affinity. In summary, OXA-48 has a very stable dimer interface, facilitated by noncovalent and predominantly charged interactions, which is stronger than the dimer interfaces previously described for other class D ß-lactamases. PDB CODES: The oxacillinase-48 (OXA-48) R206A structure has PDB ID: 5OFT and OXA-48 R189A has PDB ID: 6GOA.

Disruption of Rhodopsin Dimerization in Mouse Rod Photoreceptors by Synthetic Peptides Targeting Dimer Interface.

Synthetic peptides derived from transmembrane segments of G protein-coupled receptors (GPCR) are used to disrupt GPCR dimer interface. This peptide competition technique is an effective approach to map the dimer interface of GPCR and its functional significance. Here we present a technique to deliver synthetic transmembrane peptides to living mouse rod photoreceptors to disrupt rhodopsin (a prototypical member of Class A GPCRs) dimer formation in the endoplasmic reticulum (ER). We have shown that rhodopsin helix H1- or H8-peptide caused mislocalization of rhodopsin to the perinuclear endoplasmic reticulum (ER).