Wild-type Affinity Research Articles

Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities.

The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl. Optimal TAM signaling requires coincident TAM ligand engagement of both its receptor and the phospholipid phosphatidylserine (PtdSer): Gas6 lacking its PtdSer-binding 'Gla domain' is significantly weakened as a Tyro3/Mer agonist and is inert as an Axl agonist, even though it binds to Axl with wild-type affinity. In two settings of TAM-dependent homeostatic phagocytosis, Mer plays a predominant role while Axl is dispensable, and activation of Mer by Protein S is sufficient to drive phagocytosis.

Protease-resistant single-domain antibodies inhibit Campylobacter jejuni motility.

Camelid heavy-chain antibody variable domains (VHHs) are emerging as potential antimicrobial reagents. We have engineered a previously isolated VHH (FlagV1M), which binds Campylobacter jejuni flagella, for greater thermal and proteolytic stability. Mutants of FlagV1M were obtained from an error-prone polymerase chain reaction library that was panned in the presence of gastrointestinal (GI) proteases. Additional FlagV1M mutants were obtained through disulfide-bond engineering. Each approach produced VHHs with enhanced thermal stability and protease resistance. When the beneficial mutations from both approaches were combined, a hyperstabilized VHH was created with superior stability. The hyperstabilized VHH bound C. jejuni flagella with wild-type affinity and was capable of potently inhibiting C. jejuni motility in assays performed after sequential digestion with three major GI proteases, demonstrating the remarkable stability imparted to the VHH by combining our engineering approaches.

Enhanced stabilization of a stable single domain antibody for SEB toxin by random mutagenesis and stringent selection

Single domain antibodies, recombinant variable heavy domains derived from the unique heavy-chain only antibodies found in camelids and sharks, are exceptionally rugged due to their ability to refold following heat or chemical denaturation. In addition, a number of single domain antibodies have been found to possess high melting points which provide an even greater degree of stability; one of these, llama-derived A3, is a binder of Staphylococcal enterotoxin B and has a Tm of 83.5 °C. In this work, we utilized random mutagenesis and stringent selection in an effort to obtain variants of A3 with even higher melting points. This effort resulted in the selection of a double mutant, A3-T28I-S72I, which has a melting point of 90.0 °C and near wild-type affinity for the target antigen. We further characterized the mutations individually to determine that while both contributed to the thermal stabilization, the T28I mutation accounted for ∼ 4.1 °C of the 6.5 °C increase. This work demonstrates that by the addition of relatively subtle changes it is possible to further improve the melting temperature of single domain antibodies that are already remarkably stable.

Nicotinamide Adenine Dinucleotide-induced Multimerization of the Co-repressor CtBP1 Relies on a Switching Tryptophan

The transcriptional co-repressor C-terminal binding protein (CtBP) interacts with a number of repressor proteins and chromatin modifying enzymes. How the biochemical properties including binding of dinucleotide, oligomerization, and dehydrogenase domains of CtBP1 direct the assembly of a functional co-repressor to influence gene expression is not well understood. In the current study we demonstrate that CtBP1 assembles into a tetramer in a NAD(H)-dependent manner, proceeding through a dimeric intermediate. We find that NAD-dependent oligomerization correlates with NAD(+) binding affinity and that the carboxyl terminus is required for assembly of a dimer of dimers. Mutant CtBP1 proteins that abrogate dinucleotide-binding retain wild type affinity for the PXDLS motif, but do not self-associate either in vitro or in vivo. CtBP1 proteins with mutations in the dehydrogenase domain still retain the ability to self-associate and bind target proteins. Both co-immunoprecipitation and mammalian two-hybrid experiments demonstrate that CtBP1 self-association occurs within the nucleus, and depends on dinucleotide binding. Repression of transcription does not depend on dinucleotide binding or an intact dehydrogenase domain, but rather depends on the amino-terminal domain that recruits PXDLS containing targets. We show that tryptophan 318 (Trp(318)) is a critical residue for tetramer assembly and likely functions as a switch for effective dimerization following NAD(+) binding. These results suggest that dinucleotide binding permits CtBP1 to form an intranuclear homodimer through a Trp(318) switch, creating a nucleation site for multimerization through the C-terminal domain for tetramerization to form an effective repression complex.

The effect of ligand affinity on integrins’ lateral diffusion in cultured cells

The role of ligand affinity in altering αPS2CβPS integrins' lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CβPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22% and had a 4× slower diffusion coefficient for αPS2CβPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CβPS-V409D integrins' increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75% slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CβPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99% for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.

MHC-class I-restricted CD4 T cells: a nanomolar affinity TCR has improved anti-tumor efficacy in vivo compared to the micromolar wild-type TCR

Clinical studies with immunotherapies for cancer, including adoptive cell transfers of T cells, have shown promising results. It is now widely believed that recruitment of CD4(+) helper T cells to the tumor would be favorable, as CD4(+) cells play a pivotal role in cytokine secretion as well as promoting the survival, proliferation, and effector functions of tumor-specific CD8(+) cytotoxic T lymphocytes. Genetically engineered high-affinity T-cell receptors (TCRs) can be introduced into CD4(+) helper T cells to redirect them to recognize MHC-class I-restricted antigens, but it is not clear what affinity of the TCR will be optimal in this approach. Here, we show that CD4(+) T cells expressing a high-affinity TCR (nanomolar K (d) value) against a class I tumor antigen mediated more effective tumor treatment than the wild-type affinity TCR (micromolar K (d) value). High-affinity TCRs in CD4(+) cells resulted in enhanced survival and long-term persistence of effector memory T cells in a melanoma tumor model. The results suggest that TCRs with nanomolar affinity could be advantageous for tumor targeting when expressed in CD4(+) T cells.

Target binding to S100B reduces dynamic properties and increases Ca(2+)-binding affinity for wild type and EF-hand mutant proteins.

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca(2+) binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With (D63N)S100B as an exception ((D63N)K(D)=50±9 μM), Ca(2+) binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 ((D61N)K(D)=412±67 μM, (D65N)K(D)=968±171 μM, and (E72A)K(D)=471±133 μM), when compared to wild-type protein ((WT)K(D)=56±9 μM). For the TRTK-12 complexes, the Ca(2+)-binding affinity to wild type ((WT+TRTK)K(D)=12±10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target ((D61N+TRTK)K(D)=29±1.2 μM, (D63N+TRTK)K(D)=10±2.2 μM, (D65N+TRTK)K(D)=73±4.4 μM, and (E72A+TRTK)K(D)=18±3.7 μM). In addition, R(ex), as measured using relaxation dispersion for side-chain (15)N resonances of Asn63 ((D63N)S100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, (D61N)S100B, (D63N)S100B, and (D65N)S100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca(2+)-bound (2.0Å) and TRTK-bound (1.2Å) (D63N)S100B showed no change in Ca(2+) coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca(2+)-binding affinity in solution. Therefore, a model for how S100B-TRTK-12 complex formation increases Ca(2+) binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.

The Effect of Hydrophobic Match on Transmembrane Protein Raft Affinity

It is believed that lipid rafts control protein-protein and lipid-protein interactions within cell membranes. However, how transmembrane (TM) proteins associate with rafts remains poorly understood. One proposed mechanism is that hydrophobic match/mismatch drives TM proteins into or out of rafts. We tested this hypothesis using perfringolysin O (PFO), a member of the cholesterol-dependent cytolysin family. Upon interaction with cholesterol, PFO inserts into membranes and forms a rigid TM beta-barrel. PFO interacts with rafts, so is a useful model protein to study the origin of TM protein raft affinity. PFO mutants with different length TM segments were generated with each β-strand in each of the two TM β-hairpins lengthened (l -PFO) or shortened (s-PFO) by two residues. Similar to wildtype PFO, these mutants showed cholesterol-dependent membrane binding and pore-formation activities, although with somewhat different properties. A pore detection assay carried out to determine if the pore-forming efficiency of PFO mutants was correlated to membrane width found that l-PFO shows higher pore formation activity in vesicles with thicker bilayers than did s-PFO. Microscopy studies in giant unilamellar vesicles confirmed that both PFO mutants could bind to vesicles with co-existing Lo/Ld domains: l-PFO tends to locate within thick Lo domains, while s-PFO appears at thin Ld domains. Energy transfer (FRET) experiments were also used to compare the raft affinity of wildtype and mutated PFO in vesicles containing ordered domains. Preliminary studies indicate in cholesterol-containing vesicles having wide Lo domains (distearoylphosphatidylcholine) and thin Ld domains (dimyristoleoylphosphatidylcholine), l-PFO showed higher raft affinity, whereas s-PFO shows little raft affinity. The raft affinity of l-PFO and s-PFO was reversed in vesicles containing thinner Lo domains (dipalmitoylphosphatidylcholine/dimyristoylphosphatidylcholine) and thick Ld domains (dieicosenoylphosphatidylcholine). These studies suggest that hydrophobic match may have an important role in TM protein raft affinity.

Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins

Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na(+)-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581(T), and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former.

A combinatorial histidine scanning library approach to engineer highly pH‐dependent protein switches

There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein-protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pK(a) change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novel combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine "hot-spots," which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications.

Two Functional S100A4 Monomers Are Necessary for Regulating Nonmuscle Myosin-IIA and HCT116 Cell Invasion

S100A4, a member of the Ca(2+)-activated S100 protein family, regulates the motility and invasiveness of cancer cells. Moreover, high S100A4 expression levels correlate with poor patient survival in several cancers. Although biochemical, biophysical, and structural data indicate that S100A4 is a noncovalent dimer, it is unknown if two functional S100A4 monomers are required for the productive recognition of protein targets and the promotion of cell invasion. To address this question, we created covalently linked S100A4 dimers using a glycine rich flexible linker. The single-chain S100A4 (sc-S100A4) proteins exhibited wild-type affinities for calcium and nonmuscle myosin-IIA, retained the ability to regulate nonmuscle myosin-IIA assembly, and promoted tumor cell invasion when expressed in S100A4-deficient colon carcinoma cells. Mutation of the two calcium-binding EF-hands in one monomer, while leaving the other monomer intact, caused a 30-60-fold reduction in binding affinity for nonmuscle myosin-IIA concomitant with a weakened ability to regulate the monomer-polymer equilibrium of nonmuscle myosin-IIA. Moreover, sc-S100A4 proteins with one monomer deficient in calcium responsiveness did not support S100A4-mediated colon carcinoma cell invasion. Cross-linking and titration data indicate that the S100A4 dimer binds a single myosin-IIA target peptide. These data are consistent with a model in which a single peptide forms interactions in the vicinity of the canonical target binding cleft of each monomer in such a manner that both target binding sites are required for the efficient interaction with myosin-IIA.

A Combinatorial Approach to Engineering a Dual-Specific Metal Switch Antibody

There is considerable interest in understanding how multiple binding events can be mediated through a single protein interface. Here, a synthetic library approach was developed to generate a novel dual-specific antibody. Using a combinatorial histidine-scanning phage display library, potential metal binding sites were introduced throughout an anti-RNase A antibody interface. Stepwise selection of RNase A and metal binding produced a dual-specific antibody that retained near wild-type affinity for its target antigen while acquiring a competitive metal binding site that is capable of controlling the antibody-antigen interaction. Structure analysis of the original antibody-RNase A complex suggested peripheral interface residues and loop flexibility are key contributors for obtaining the dual specificity.

Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2–7 Complex, Allowing the GINS Tetramer to Bind the Mcm2–7 Complex

The replication fork helicase in eukaryotic cells is comprised of Cdc45, Mcm2-7, and GINS (CMG complex). In budding yeast, Sld3, Sld2, and Dpb11 are required for the initiation of DNA replication, but Sld3 and Dpb11 do not travel with the replication fork. Sld3 and Cdc45 bind to early replication origins during the G(1) phase of the cell cycle, whereas Sld2, GINS, polymerase ε, and Dpb11 form a transient preloading complex that associates with origins during S phase. We show here that Sld3 binds tightly to origin single-stranded DNA (ssDNA). CDK-phosphorylated Sld3 binds to origin ssDNA with similar high affinity. Origin ssDNA does not disrupt the interaction between Sld3 and Dpb11, and origin ssDNA does not disrupt the interaction between Sld3 and Cdc45. However, origin ssDNA substantially disrupts the interaction between Sld3 and Mcm2-7. GINS and Sld3 compete with one another for binding to Mcm2-7. However, in a mixture of Sld3, GINS, and Mcm2-7, origin ssDNA inhibits the interaction between Sld3 and Mcm2-7, whereas origin ssDNA promotes the association between GINS and Mcm2-7. We also show that origin single-stranded DNA promotes the formation of the CMG complex. We conclude that origin single-stranded DNA releases Sld3 from Mcm2-7, allowing GINS to bind Mcm2-7.

Abstract 3265A: GIST-associated c-Kit mutants exhibit differential affinities to hsp90 and its co-chaperone Cdc37

Abstract Background: Hsp90 inhibitors have been introduced to clinical treatment of imatinib-resistant GIST, but their efficacy is dependant on KIT mutations. We analysed the role of the chaperone Hsp90 as stabilizing factor for wild-type and oncogenic, misfolded c-Kit. Methods: the intracellular part of wild-type and mutated c-Kit (aa544-977) fused with an N-terminal TAP-tag was expressed in HEK293T cells. The clinically most abundant primary and imatinib-resistant secondary c-Kit mutations were selected: V560D; V559D; del557,558; L576P; K642E; V560D+D820A; del557,558+Y823D; D820A. A shortened TAP assay followed by LC-MS/MS was applied for identification of interaction partners. For verification a GST pulldown assay was used. Relative quantifications of mutation-specific interaction partners were performed by quantitative immunoblotting using an antibody raised against the C-terminus of human c-Kit. In addition, two GIST cell lines containing an imatinib-resistant and an imatinib-sensitive c-Kit mutation were investigated by quantitative immunoblotting. The activityof hsp90 inhibitor 17-AAG was studied in whole cell lysates of HEK293T cells expressing selected mutated c-Kit constructs. Results: Using the TAP-c-Kit constructs for a shortened TAP assay, Hsp90 and its cochaperone Cdc37 were identified as specific interaction partners for the GIST-associated c-Kit mutants. This interaction was confirmed using GST-Cdc37 pulldown analysis. Differential analysis of c Kit/Hsp90 interaction revealed mutation-specific affinities (up to a relative factor of about 8), with D820A showing the lowest, close-to wild-type affinity, and L576P showing the highest affinity. Among the two GIST cell lines studied, the imatinib-resistant mutant (c-Kit K642E) exhibited a 2.5 fold higher affinity to Hsp90 compared to the imatinib-sensitive mutant (c-Kit V560D+D820A). In both cell lines, the Cdc37 and Hsp90 affinities are observed in parallel. A closely related situation was observed for the interactions of the corresponding TAP-c-Kit constructs. In addition, the influence of the When analysing hsp90 inhibitor 17AAG (17-(allylamino)-17-demethoxygeldanamycin)) incells expressing selected mutated c-Kit constructs, western blot analyses showed significant degradation of total c-Kit relative to untreated controls in all mutants studied. Amongst these, mutant D820A was found to be less sensitive to 17AAG. Wild-type c-Kit showed no sensitivity against 17AAG. Conclusion: this study demonstrates mutation-specific affinities between Hsp90 and Cdc37 with c-Kit mutants. It also points to a conformity between this phenomenon and the 17AAG effect. These results allow a better understanding of the potential of hsp90 inhibition in GIST therapy and better selection of candidates for treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3265A. doi:10.1158/1538-7445.AM2011-3265A

Genetic Screen to Isolate “Lariat” Peptides for Characterizing ABL Kinase Activity and Conformation

AbstractAbstract 438Type II Abl kinase inhibitors such as imatinib achieve high selectivity by specifically targeting the inactive Abl/c-Kit conformation. However, these inhibitors are prone to inactivation by resistance mutations that cause conformational changes. A better understanding of mutant Abl conformations would provide information to design inhibitors that recognize multiple states of Abl with lower rates of resistance. To more easily examine Abl kinase conformations, conformer specific reagents are needed that are (i) easily generated against a given Abl conformation, (ii) compatible with genetic selections, in vivo and in vitro assays, and structural studies, and (iii) amenable to chemical synthesis. Recently, we developed a novel genetic screen for isolating “lariat” peptide inhibitors of protein function. Lariats are lactone-cyclized peptides that possess the above-mentioned characteristics and can be used to evaluate the function and therapeutic potential of proteins. Previously, we isolated specific lariat inhibitors against the inactive conformation of the bacterial repressor LexA. To obtain conformer specific lariats against Abl, we screened a combinatorial seven amino acid lariat library for interactions with the Abl SH1 domain using the yeast two-hybrid (Y2H) assay. We obtained two lariat peptides named A1 (SGWQRLPFEY) and A2 (SGWHRLSEEY) that interacted with the Abl SH1 domain. In vitro studies with purified Abl kinase demonstrated that the A1 lariat competitively inhibited ATP binding with an inhibitory constant of 5.95 μM. By performing site-saturation mutagenesis, we defined acceptable and tolerable substitutions at each position of the A1 lariat. To obtain tighter binding variants of the A1 lariat, we rationally designed mutations and constructed a second-generation lariat (GWQTLDWNY) with | 10 times higher affinity for Abl. We compared the affinity of the lariats for imatinib resistant Abl kinase mutants using the lariat Y2H binding assay. Mutations that (i) promote conformational dynamics of the kinase T315I), (ii) destabilize the Abl/c-Kit like inactive state (H396R), (iii) disrupt the flexibility or hydrophobicity of the lariat-binding pocket (E355G), and (iv) distort the ATP phosphate binding loop (Y253F), have a marked decrease in lariat affinity. Together, these results show that the lariat preferentially bound to the Abl/c-Kit like inactive conformation and act similarly to type II kinase inhibitors. A specificity screen against a panel of related and distant kinases also demonstrated the conformer specific nature of the lariats. We used lariats that recognized the Abl/c-Kit like inactive state to probe various conformations of Abl His396 mutants. His396 is located in the activation loop and does not alter the Abl/c-Kit like inactive form. His396Pro and His396Arg substitutions are clinically relevant imatinib resistant mutations that alter the conformation of Abl. Previous structural studies showed that the His396Pro mutation causes Abl to adopt the active conformation. It is hypothesized that the His396Arg mutation favors the inactive Src/Cdk like state. Using inactive conformation specific Abl lariats as probes, we showed that these lariats interact weakly with the His396Arg relative to His396, suggesting that this mutation destabilized the Abl/c-Kit like inactive state. We also showed that the lariats interacted stronger with a previously unreported His396Ile mutation than His396, indicating that His396Ile shifts Abl to a highly inactive conformation. Preliminary studies suggest that this residue plays a key role in determining the flexibility of the activation loop and hence the conformational state of Abl. These results provide information on the structure and sequence requirements for maintaining an auto-inhibited activation loop conformation. In summary, we isolated lariat peptide inhibitors against the Abl SH1 domain, characterized their mechanism of action, and improved their affinity for wild type and mutant Abl SH1domains. Additionally, we demonstrated that lariat peptides can be used as affinity reagents to probe conformational states of Abl. Disclosures:No relevant conflicts of interest to declare.

Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder

Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (Tm) of 39.3±1.0 and 37.1±0.7°C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35°C over 120min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (ΔTmmax=8°C, half-maximal effective ligand concentration, AC50=3μM). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in Tm of ∼16°C, followed by MeCbl at ∼13°C, each evaluated at 50μM cofactor. The other cobalamins stabilized in the order (CN)2Cbi>OHCbl>CNCbl. Interestingly, the AC50’s for AdoCbl, MeCbl, (CN)2Cbi and OHCbl were similar and ranged from 1–3μM, which compares well with the Kd of 6μM for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (ΔTmmax=4°C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC50 of 7μM. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC50 for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease.

Computational design of second‐site suppressor mutations at protein–protein interfaces

The importance of a protein-protein interaction to a signaling pathway can be established by showing that amino acid mutations that weaken the interaction disrupt signaling, and that additional mutations that rescue the interaction recover signaling. Identifying rescue mutations, often referred to as second-site suppressor mutations, controls against scenarios in which the initial deleterious mutation inactivates the protein or disrupts alternative protein-protein interactions. Here, we test a structure-based protocol for identifying second-site suppressor mutations that is based on a strategy previously described by Kortemme and Baker. The molecular modeling software Rosetta is used to scan an interface for point mutations that are predicted to weaken binding but can be rescued by mutations on the partner protein. The protocol typically identifies three types of specificity switches: knob-in-to-hole redesigns, switching hydrophobic interactions to hydrogen bond interactions, and replacing polar interactions with nonpolar interactions. Computational predictions were tested with two separate protein complexes; the G-protein Galpha(i1) bound to the RGS14 GoLoco motif, and UbcH7 bound to the ubiquitin ligase E6AP. Eight designs were experimentally tested. Swapping a buried hydrophobic residue with a polar residue dramatically weakened binding affinities. In none of these cases were we able to identify compensating mutations that returned binding to wild-type affinity, highlighting the challenges inherent in designing buried hydrogen bond networks. The strongest specificity switches were a knob-in-to-hole design (20-fold) and the replacement of a charge-charge interaction with nonpolar interactions (55-fold). In two cases, specificity was further tuned by including mutations distant from the initial design. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

A Pit-1 Threonine 220 Phosphomimic Reduces Binding to Monomeric DNA Sites to Inhibit Ras and Estrogen Stimulation of the Prolactin Gene Promoter

Pit-1 is a POU-homeodomain transcription factor that dictates the ontogeny of pituitary somatotrophs, lactotrophs, and thyrotrophs through regulation of their respective protein hormone genes: GH, prolactin (PRL), and TSHbeta. Although Pit-1 threonine 220 (T220) and serine 115 are protein kinase phospho-acceptor sites, the transcriptional role of Pit-1 phosphorylation remains unclear. In the rat PRL promoter (rPRL), Ras-stimulated transcription is mediated by binding of Ets-1 and Pit-1 at a composite site (FPIV). Ets-1 and Pit-1 physically interact, and Pit-1 T220 is a major Ets-1 contact point. T220 was mutated to aspartic acid (D, to mimic phosphorylation) or a neutral alanine (A), and DNA binding and transcriptional activity were tested. The Pit-1 T220D mutation reduced binding at monomeric Pit-1 sites (FPIV, PRL-1d), but not dimeric Pit-1 sites (FPI). Pit-1 T220A bound all sites with wild-type (WT) affinity. In transfections of HeLa cells, each Pit-1 mutant transcriptionally activated the -425rPRL promoter and cooperated with Ets-1 to WT levels. In contrast, Pit-1-mediated Ras activation of the -425 rPRL promoter was significantly inhibited by T220D. Finally, Pit-1 synergistic activation of the 2500-bp rPRL promoter with estrogen receptor was reduced by T220D compared with T220A and WT Pit-1. Thus, phosphorylation of Pit-1 T220 reduces binding to monomeric sites blunting Ras and estrogen/estrogen receptor stimulation of the rPRL gene promoter. Consequently, T220 phosphorylation of Pit-1 by protein kinase A, protein kinase C, or cell cycle-dependent kinases appears to serve as a regulatory switch, inhibiting Ras and estrogen/estrogen receptor regulatory pathways, while enhancing the cAMP/protein kinase A response, thus allowing a more precise integration of pituitary responses to distinct signaling stimuli.

Mechanism of vitamin B 12-responsiveness in cblC methylmalonic aciduria with homocystinuria

Patients with the cblC vitamin B 12 (cobalamin, cbl) disorder are defective in the intracellular synthesis of adenosylcobalamin and methylcobalamin and have combined homocystinuria and methylmalonic aciduria. While other vitamin B 12 disorders are treatable with high dose cyanocobalamin (CNCbl) or hydroxocobalamin (OHCbl), cblC patients respond well to OHCbl but not to CNCbl. Patient mutations were introduced into recombinant MMACHC ( cblC) protein and the binding of CNCbl and OHCbl was examined. Three mutations were analyzed: G147D, associated with early onset, vitamin B 12 unresponsive disease; R161Q, associated with late onset disease that is highly responsive to OHCbl; and H122A, selected to test the hypothesis that H122 is central to a proposed vitamin B 12 binding motif on MMACHC. We report here that wild-type MMACHC binds both OHCbl and CNCbl with similar, tight affinity ( K d = 5.7 μM). We also report that MMACHC binds CNCbl in the base-off form, with the dimethylbenzimidazole (DMB) base of cobalamin displaced from coordination with the cobalt. In this form, wild-type MMACHC is able to reductively decyanate CNCbl to cob(II)alamin requiring only the presence of NADPH and FAD. We demonstrate that MMACHC with the G147D mutation is unable to bind either CNCbl or OHCbl, providing a straight forward explanation for the absence of response to either vitamin form. However, we show that MMACHC containing the R161Q mutation binds OHCbl with wild-type affinity, but is disturbed in binding CNCbl and has impaired decyanation. Finally, we show that H122A has reduced binding, but like R161Q, it binds OHCbl more tightly than CNCbl, suggesting that this histidine is not absolutely required for binding. These studies suggest that the ability of mutant MMACHC to respond to vitamin therapy depends on its ability to bind the vitamin with significant affinity, and for CNCbl, also on its ability to bind in the base-off form to facilitate reductive decyanation. These studies emphasize the continued use of OHCbl with cblC patients for maximum therapeutic effect.

Analysis of a Critical Interaction within the Archaeal Box C/D Small Ribonucleoprotein Complex

In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are responsible for 2'-O-methylation of tRNAs and rRNAs. The archaeal box C/D small RNP complex requires a small RNA component (sRNA) possessing Watson-Crick complementarity to the target RNA along with three proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from S-adenosylmethionine to the target RNA is performed by fibrillarin, which by itself has no affinity for the sRNA-target duplex. Instead, it is targeted to the site of methylation through association with Nop5p, which in turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a bridge between the targeting and catalytic functions of the box C/D small RNP complex, we have employed alanine scanning to evaluate the interaction between the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA complex. From these data, we were able to construct an isolated RNA-binding domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography and binds to the L7Ae box C/D RNA complex with near wild type affinity. These data demonstrate that the Nop-RBD is an autonomously folding and functional module important for protein assembly in a number of complexes centered on the L7Ae-kinkturn RNP.