Society of Radiologists in Ultrasound Consensus on Routine Pelvic US for Endometriosis.

Endometriosis is a prevalent and potentially debilitating condition that mostly affects individuals of reproductive age, and often has a substantial diagnostic delay. US is usually the first-line imaging modality used when patients report chronic pelvic pain or have issues of infertility, both common symptoms of endometriosis. Other than the visualization of an endometrioma, sonologists frequently do not appreciate endometriosis on routine transvaginal US images. Given a substantial body of literature describing techniques to depict endometriosis at US, the Society of Radiologists in Ultrasound convened a multidisciplinary panel of experts to make recommendations aimed at improving the screening process for endometriosis. The panel was composed of experts in the imaging and management of endometriosis, including radiologists, sonographers, gynecologists, reproductive endocrinologists, and minimally invasive gynecologic surgeons. A comprehensive literature review combined with a modified Delphi technique achieved a consensus. This statement defines the targeted screening population, describes techniques for augmenting pelvic US, establishes direct and indirect observations for endometriosis at US, creates an observational grading and reporting system, and makes recommendations for additional imaging and patient management. The panel recommends transvaginal US of the posterior compartment, observation of the relative positioning of the uterus and ovaries, and the uterine sliding sign maneuver to improve the detection of endometriosis. These additional techniques can be performed in 5 minutes or less and could ultimately decrease the delay of an endometriosis diagnosis in at-risk patients.

CFD-based bioreactor model with proportional-integral-derivative controller functionality for dissolved oxygen and pH.

A physics-based model for predicting cell culture fluid properties inside a stirred tank bioreactor with embedded PID controller logic is presented. The model evokes a time-accurate solution to the fluid velocity field and overall volumetric mass transfer coefficient, as well as the ongoing effects of interfacial mass transfer, species mixing, and aqueous chemical reactions. The modeled system also includes a direct coupling between process variables and system control variables via embedded controller logic. Satisfactory agreement is realized between the model prediction and measured bioreactor data in terms of the steady-state operating conditions and the response to setpoint changes. Simulation runtimes are suitable for industrial research and design timescales.

Allosteric regulation within the highly interconnected structural scaffold of AraC/XylS homologs tolerates a wide range of amino acid changes.

To create bacterial transcription "circuits" for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coli AraC transcriptional activator, for which binding to DNA is modulated by binding L-arabinose. Here, we engineered chimeric AraC/XylS transcription activators that recognized ara DNA binding sites and responded to varied effector ligands. The first step, identifying domain boundaries in the natural homologs, was challenging because (i) no full-length, dimeric structures were available and (ii) extremely low sequence identities (≤10%) among homologs precluded traditional assemblies of sequence alignments. Thus, to identify domains, we built and aligned structural models of the natural proteins. The designed chimeric activators were assessed for function, which was then further improved by random mutagenesis. Several mutational variants were identified for an XylS•AraC chimera that responded to benzoate; two enhanced activation to near that of wild-type AraC. For an RhaR•AraC chimera, a variant with five additional substitutions enabled transcriptional activation in response to rhamnose. These five changes were dispersed across the protein structure, and combinatorial experiments testing subsets of substitutions showed significant non-additivity. Combined, the structure modeling and epistasis suggest that the common AraC/XylS structural scaffold is highly interconnected, with complex intra-protein and inter-domain communication pathways enabling allosteric regulation. At the same time, the observed epistasis and the low sequence identities of the natural homologs suggest that the structural scaffold and function of transcriptional regulation are nevertheless highly accommodating of amino acid changes.

Untargeted proteomics reveals upregulation of stress response pathways during CHO-based monoclonal antibody manufacturing process leading to disulfide bond reduction.

Monoclonal antibody (mAb) interchain disulfide bond reduction can cause a loss of function and negatively impact the therapeutic's efficacy and safety. Disulfide bond reduction has been observed at various stages during the manufacturing process, including processing of the harvested material. The factors and mechanisms driving this phenomenon are not fully understood. In this study, we examined the host cell proteome as a potential factor affecting the susceptibility of a mAb to disulfide bond reduction in the harvested cell culture fluid (HCCF). We used untargeted liquid-chromatography-mass spectrometry-based proteomics experiments in conjunction with a semi-automated protein identification workflow to systematically compare Chinese hamster ovary (CHO) cell protein abundances between bioreactor conditions that result in reduction-susceptible and reduction-free HCCF. Although the growth profiles and antibody titers of these two bioreactor conditions were indistinguishable, we observed broad differences in host cell protein (HCP) expression. We found significant differences in the abundance of glycolytic enzymes, key protein reductases, and antioxidant defense enzymes. Multivariate analysis of the proteomics data determined that upregulation of stress-inducible endoplasmic reticulum (ER) and other chaperone proteins is a discriminatory characteristic of reduction-susceptible HCP profiles. Overall, these results suggest that stress response pathways activated during bioreactor culture increase the reduction-susceptibility of HCCF. Consequently, these pathways could be valuable targets for optimizing culture conditions to improve protein quality.

Predictors for Discontinuation of Intrauterine Devices That Underwent Ultrasound-Guided Placement in a Long-term Follow-up Cohort.

OBJECTIVES: To investigate long-term outcomes of ultrasound-guided intrauterine device (IUD) insertion. The rate of and reasons for IUD discontinuation were reviewed, including the prevalence of uterine fibroids. METHODS: A retrospective cohort of patients who underwent transabdominal ultrasound (TAUS)-guided IUD insertion was reviewed. Information collected included age, body mass index, insertion date, indication for IUD use, indication for using TAUS guidance, and presence of uterine fibroids. The date of and reason for discontinuation were recorded. RESULTS: One hundred sixteen patients with successful TAUS-guided IUD placement were identified. Of these, 51 patients (43.97%) no longer had an IUD in place. An overall actual-to-expected use ratio was calculated to be 63.49%. The most common reason for IUD discontinuation was spontaneous expulsion (11.21%). The prevalence of fibroid uteri was 27.6% in our cohort. The actual-to-expected use ratio of those with a fibroid uterus was calculated to be 43.28%, which was significantly lower than that for a nonfibroid uterus (73.80%; P = .002). There were 27 of 104 patients with IUD use of less than 1 year, and fibroids were present 44.4% of the time. Comparatively, of the 77 patients that had IUD continuation of greater than 1 year, only 24.7% had fibroids (P = .022). The yearly continuation rates over 5 years were 74.04%, 55.84%, 41.67%, 35.14%, and 32.0% respectively. Of the 18 patients who received TAUS-guided insertion for a previous IUD expulsion, 33.3% had another spontaneous expulsion. CONCLUSIONS: Uterine fibroids and a previous expulsion appear to be the most likely predictors of IUD discontinuation, particularly within 1 year after insertion.

Increased MSX level improves biological productivity and production stability in multiple recombinant GS CHO cell lines.

Increasing cell culture productivity of recombinant proteins via process improvements is the primary focus for research groups within biologics manufacturing. Any recommendations to improve a manufacturing process obviously must be effective, but also be robust, scalable, and with product quality comparable to the original process. In this study, we report that three different GS-/- CHO cell lines developed in media containing a standard concentration of the selection agent methionine sulfoximine (MSX), but then exposed to increased MSX concentrations during seed train expansion, achieved titer increases of 10-19%. This result was observed in processes already considerably optimized. Expanding the cells with a higher MSX concentration improved cell line production stability with increased culture age. Production cultures in 500-L and 1000-L bioreactors replicated laboratory results using 5-L bioreactors, demonstrating process robustness and scalability. Furthermore, product quality attributes of the final drug substance using the higher MSX process were comparable with those from cells expanded in media with the standard selection MSX concentration. Subsequent mechanistic investigations confirmed that the cells were not altered at the genetic level in terms of integration profiles or gene copy number, nor transcriptional levels of glutamine synthetase, heavy chain, or light chain genes. This study provides an effective and applicable strategy to improve the productivity of therapeutic proteins for biologics manufacturing.

Metabolic understanding of disulfide reduction during monoclonal antibody production.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract.

Dyspareunia Related to GSM: Association of Total Vaginal Thickness via Transabdominal Ultrasound.

INTRODUCTION: It has previously been suggested in the literature that ultrasound measurement of total vaginal wall thickness (TVT) differs significantly between pre- and postmenopausal women, indicating that it may be a useful and noninvasive objective assessment to correlate the degree of vaginal atrophy to patient-reported symptoms. AIM: The purpose of this cross-sectional pilot study was to determine whether TVT in postmenopausal women, as measured with transabdominal ultrasound, is associated with patient-reported dyspareunia and symptoms related to genitourinary symptomatology. METHODS: Postmenopausal women presenting for pelvic ultrasound had TVT and total mucosal thickness (TMT) measured via transabdominal ultrasound. A questionnaire also was administered assessing menopausal status, relevant medical history, and self-report of dyspareunia and other symptoms related to the genitourinary syndrome of menopause (GSM). This questionnaire was derived from the Vulvovaginal Symptom Questionnaire, which has been validated in the literature. MAIN OUTCOME MEASURE: The main outcome measures included the average TVT and TMT for postmenopausal women reporting any symptom of GSM and average TVT and TMT of women reporting no symptoms of GSM. RESULTS: Data from 44 postmenopausal women showed no significant association between transabdominal ultrasound-measured TVT or TMT and patient report of dyspareunia or other genitourinary symptoms. Data were stratified by individual GSM symptoms, sexual symptoms as an aggregate, and individual sexual symptoms. Neither of these subgroups showed a statistically significant difference in TVT or TMT between symptomatic and asymptomatic women. CLINICAL IMPLICATIONS: Although no statistically significant data were derived from this study, we propose that future studies investigating the longitudinal relationship between TVT and GSM symptomatology may show an association between total vaginal thickness measurement change over time as determined by ultrasound with the presence of patient-reported dyspareunia and other GSM symptoms. STRENGTHS & LIMITATIONS: This study is limited by its small sample size as well as the patient population, which was restricted to postmenopausal women with a clinical indication for ultrasound. A major strength of this investigation is that it is the first study to look at the relationship between sexual pain and other GSM symptoms and TVT using transabdominal ultrasound, which is a readily available, non-invasive tool in most clinical settings. CONCLUSION: Based on the results of this small pilot study, transabdominal pelvic ultrasound cannot be used at this time to objectively quantify the presence of sexual pain or other GSM symptoms; however, future studies should continue to investigate the longitudinal relationship between these 2 variables. Balica AC, Cooper AM, McKevitt MK, et al. Dyspareunia Related to GSM: Association of Total Vaginal Thickness via Transabdominal Ultrasound. J Sex Med 2019; 16:2038-2042.

Sonographically Guided Office Endometrial Sampling: Indications and Results.

OBJECTIVES: The purpose of this study was to identify clinical indications in which endometrial sampling was performed under transabdominal ultrasound (US) guidance and to evaluate the outcomes of this approach in an academic practice. METHODS: We retrieved data from the electronic medical record for all patients referred to the gynecologic US unit for transabdominal US-guided endometrial sampling from January 2011 to June 2017. Data retrieved included age, parity, previous cesarean delivery or pelvic surgery, indication for endometrial biopsy, US abnormalities, clinical reasoning for US-guided sampling, and pathologic reports. RESULTS: A total of 113 patients were referred for US-guided sampling between January 2011 and June 2017. We identified the following reasons for US-guided biopsy referral: failed blind biopsy attempt, fibroids, uterine position or anomaly, need for targeted sampling, cervical stenosis, and other indications. Ninety-five of the 113 patients (84%) underwent successful US-guided endometrial sampling. Forty of the 113 patients were referred after a failed blind biopsy, with 83% subsequently undergoing successful US-guided endometrial sampling. Of the 30 patients referred for fibroids, 29 (97%) underwent successful US-guided sampling. High success rates were also noted for transabdominal US guidance referrals for the uterine position or anomaly (86%) and the need for targeted sampling (83%). CONCLUSIONS: Our results suggest that endometrial sampling performed under US guidance could be considered for patients with a failed blind approach, fibroids, uterine anomalies, and interest in targeted sampling. In such cases, US-guided sampling could be considered before surgical options.

Sonographically guided insertion of intrauterine device: Indications and results.

From 2011 to 2015, a total of 67 patients were referred for IUD insertion guided with transabdominal sonography (TAS). Fifty-six of the 67 patients had successful IUD insertion under TAS guidance. The clinical indications for referral included fibroids, uterine position, previous history of IUD expulsion, and limited tolerance of pelvic examination. Reasons for failed TAS-guided IUD insertion included patient discomfort, cervical stenosis, and inability to remove and replace an existing device. Ultrasound guidance could help broaden the patient population that may benefit from the therapeutic value of an IUD.

Transabdominal sonography to measure the total vaginal and mucosal thicknesses.

Transabdominal and transvaginal sonography are used to measure bladder wall and detrusor thickness. Only transvaginal sonography has been used to measure the vaginal wall thickness. We describe the use of transabdominal sonography to measure the total vaginal wall thickness and total vaginal mucosal thickness at the bladder trigone. The mean bladder wall thickness and SD from published data were within the 95% confidence interval of our data. Total vaginal and mucosal thicknesses are reliable measurements, which require specific evaluation in a postmenopausal population. They could be used to quantify vaginal atrophy and could correlate to symptoms of atrophy and response to treatment. © 2017 Wiley Periodicals, Inc. J Clin Ultrasound 45:461-464, 2017.

Assessing the thickness of the vaginal wall and vaginal mucosa in pre-menopausal versus post-menopausal women by transabdominal ultrasound: A feasibility study.

OBJECTIVE: As life expectancy increases, the number of women reporting adverse genito-urinary symptoms (genitourinary syndrome of menopause; GSM) from menopause, including vaginal dryness and sexual pain, also will increase. Current objective measurements of vaginal atrophy such as maturation index require vaginal swabs and are invasive; at present, no minimally invasive measurements exist. The purpose of this study was to assess whether total vaginal wall thickness (TVT) and total vaginal mucosa thickness (TMT) as measured by transabdominal ultrasound could qualify as additional objective markers of vaginal wall thinning which could be related to menopausal status. DESIGN: Women presenting for pelvic ultrasound had a transabdominal ultrasound scan performed to measure TVT and TMT at the level of the bladder trigone. In addition, a transvaginal endometrial lining thickness was measured. RESULTS: The ultrasound measurement data from 76 participants showed that there was a significant difference in the mean value for TVT and endometrial lining between pre- and post-menopausal women. The same difference in mean was not observed for TMT. CONCLUSION: TVT may be a reliable measure of vaginal thinning, which worsens with estrogen decline. These preliminary data also suggest that TMT does not have the same correlation as the TVT measurement. A larger sample is needed to further assess the usefulness and sensitivity of these measures and whether there is clinical and/or research usefulness in obtaining vaginal wall measurements by transabdominal ultrasound.

1.65†Šresolution structure of the AraC-family transcriptional activator ToxT from Vibrio cholerae.

ToxT is an AraC-family transcriptional activator protein that controls the expression of key virulence factors in Vibrio cholerae, the causative agent of cholera. ToxT directly activates the expression of the genes that encode the toxin-coregulated pilus and cholera toxin, and also positively auto-regulates its own expression from the tcp promoter. The crystal structure of ToxT has previously been solved at 1.9 Šresolution (PDB entry 3gbg). In this study, a crystal structure of ToxT at 1.65 Šresolution with a similar overall structure to the previously determined structure is reported. However, there are distinct differences between the two structures, particularly in the region that extends from Asp101 to Glu110. This region, which can influence ToxT activity but was disordered in the previous structure, can be traced entirely in the current structure.

3-Substituted Biquinolinium Inhibitors of AraC Family Transcriptional Activator VirF from S. flexneri Obtained Through In Situ Chemical Ionization of 3,4-Disubstituted Dihydroquinolines.

During a structure-activity relationship optimization campaign to develop an inhibitor of AraC family transcriptional activators, we discovered an unexpected transformation of a previously reported inhibitor that occurs under the assay conditions. Once placed in the assay media, the 3, 4-disubstituted dihydroquinoline core of the active analogue rapidly undergoes a decomposition reaction to a quaternary 3-substituted biquinolinium. Further examination established an SAR for this chemotype while also demonstrating its resilience to irreversible binding of biologically relevant nucleophiles.

Small-molecule inhibitor of the Shigella flexneri master virulence regulator VirF.

VirF is an AraC family transcriptional activator that is required for the expression of virulence genes associated with invasion and cell-to-cell spread by Shigella flexneri, including multiple components of the type three secretion system (T3SS) machinery and effectors. We tested a small-molecule compound, SE-1 (formerly designated OSSL_051168), which we had identified as an effective inhibitor of the AraC family proteins RhaS and RhaR, for its ability to inhibit VirF. Cell-based reporter gene assays with Escherichia coli and Shigella, as well as in vitro DNA binding assays with purified VirF, demonstrated that SE-1 inhibited DNA binding and transcription activation (likely by blocking DNA binding) by VirF. Analysis of mRNA levels using real-time quantitative reverse transcription-PCR (qRT-PCR) further demonstrated that SE-1 reduced the expression of the VirF-dependent virulence genes icsA, virB, icsB, and ipaB in Shigella. We also performed eukaryotic cell invasion assays and found that SE-1 reduced invasion by Shigella. The effect of SE-1 on invasion required preincubation of Shigella with SE-1, in agreement with the hypothesis that SE-1 inhibited the expression of VirF-activated genes required for the formation of the T3SS apparatus and invasion. We found that the same concentrations of SE-1 had no detectable effects on the growth or metabolism of the bacterial cells or the eukaryotic host cells, respectively, indicating that the inhibition of invasion was not due to general toxicity. Overall, SE-1 appears to inhibit transcription activation by VirF, exhibits selectivity toward AraC family proteins, and has the potential to be developed into a novel antibacterial agent.

Identification of a small-molecule inhibitor of bacterial AraC family activators.

Protein members of the AraC family of bacterial transcriptional activators have great promise as targets for the development of novel antibacterial agents. Here, we describe an in vivo high-throughput screen to identify inhibitors of the AraC family activator protein RhaS. The screen used two Escherichia coli reporter fusions: one to identify potential RhaS inhibitors and a second to eliminate nonspecific inhibitors from consideration. One compound with excellent selectivity, OSSL_051168, was chosen for further study. OSSL_051168 inhibited in vivo transcription activation by the RhaS DNA-binding domain to the same extent as the full-length protein, indicating that this domain was the target of its inhibition. Growth curves showed that OSSL_051168 did not affect bacterial cell growth at the concentrations used in this study. In vitro DNA-binding assays with purified protein suggest that OSSL_051168 inhibits DNA binding by RhaS. In addition, we found that it inhibits DNA binding by a second AraC family protein, RhaR, which shares 30% amino acid identity with RhaS. OSSL_051168 did not have a significant impact on DNA binding by the non-AraC family proteins CRP and LacI, suggesting that the inhibition is likely specific for RhaS, RhaR, and possibly additional AraC family activator proteins.

The AraC/XylS family activator RhaS negatively autoregulates rhaSR expression by preventing cyclic AMP receptor protein activation.

The Escherichia coli RhaR protein activates expression of the rhaSR operon in the presence of its effector, L-rhamnose. The resulting RhaS protein (plus L-rhamnose) activates expression of the L-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively. Here, we further investigated our previous finding that rhaS deletion resulted in a threefold increase in rhaSR promoter activity, suggesting RhaS negative autoregulation of rhaSR. We found that RhaS autoregulation required the cyclic AMP receptor protein (CRP) binding site at rhaSR and that RhaS was able to bind to the RhaR binding site at rhaSR. In contrast to the expected repression, we found that in the absence of both RhaR and the CRP binding site at the rhaSR promoter, RhaS activated expression to a level comparable with RhaR activation of the same promoter. However, when the promoter included the RhaR and CRP binding sites, the level of activation by RhaS and CRP was much lower than that by RhaR and CRP, suggesting that CRP could not fully coactivate with RhaS. Taken together, our results indicate that RhaS negative autoregulation involves RhaS competition with RhaR for binding to the RhaR binding site at rhaSR. Although RhaS and RhaR activate rhaSR transcription to similar levels, CRP cannot effectively coactivate with RhaS. Therefore, once RhaS reaches a relatively high protein concentration, presumably sufficient to saturate the RhaS-activated promoters, there will be a decrease in rhaSR transcription. We propose a model in which differential DNA bending by RhaS and RhaR may be the basis for the difference in CRP coactivation.

Differences in the mechanism of the allosteric l-rhamnose responses of the AraC/XylS family transcription activators RhaS and RhaR.

Proteins in the largest subset of AraC/XylS family transcription activators, including RhaS and RhaR, have C-terminal domains (CTDs) that mediate DNA-binding and transcription activation, and N-terminal domains (NTDs) that mediate dimerization and effector binding. The mechanism of the allosteric effector response in this family has been identified only for AraC. Here, we investigated the mechanism by which RhaS and RhaR respond to their effector, l-rhamnose. Unlike AraC, N-terminal truncations suggested that RhaS and RhaR do not use an N-terminal arm to inhibit activity in the absence of effector. We used random mutagenesis to isolate RhaS and RhaR variants with enhanced activation in the absence of l-rhamnose. NTD substitutions largely clustered around the predicted l-rhamnose-binding pockets, suggesting that they mimic the structural outcome of effector binding to the wild-type proteins. RhaS-CTD substitutions clustered in the first HTH motif, and suggested that l-rhamnose induces improved DNA binding. In contrast, RhaR-CTD substitutions clustered at a single residue in the second HTH motif, at a position consistent with improved RNAP contacts. We propose separate allosteric mechanisms for the two proteins: Without l-rhamnose, RhaS does not effectively bind DNA while RhaR does not effectively contact RNAP. Upon l-rhamnose binding, both proteins undergo structural changes that enable transcription activation.

Transcription activation by the DNA-binding domain of the AraC family protein RhaS in the absence of its effector-binding domain.

The Escherichia coli L-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of L-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of L-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a DeltarhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.

Functional consequences of exchanging domains between LacI and PurR are mediated by the intervening linker sequence.

Homologue function can be differentiated by changing residues that affect binding sites or long-range interactions. LacI and PurR are two proteins that represent the LacI/GalR family (>500 members) of bacterial transcription regulators. All members have distinct DNA-binding and regulatory domains linked by approximately 18 amino acids. Each homologue has specificity for different DNA and regulatory effector ligands; LacI and PurR also exhibit differences in allosteric communication between DNA and effector binding sites. A comparative study of LacI and PurR suggested that alterations in the interface between the regulatory domain and linker are important for differentiating their functions. Four residues (equivalent to LacI positions 48, 55, 58, and 61) appear particularly important for creating a unique interface and were predicted to be necessary for allosteric regulation. However, nearby residues in the linker interact with DNA ligand. Thus, differences observed in interactions between linker and regulatory domain may be the cause of altered function or an effect of the two proteins binding different DNA ligands. To separate these possibilities, we created a chimeric protein with the LacI DNA-binding domain/linker and the PurR regulatory domain (LLhP). If the interface requires homologue-specific interactions in order to propagate the signal from effector binding, then LLhP repression should not be allosterically regulated by effector binding. Experiments show that LLhP is capable of repression from lacO1 and, contrary to expectation, allosteric response is intact. Further, restoring the potential for PurR-like interactions via substitutions in the LLhP linker tends to diminish repression. These effects are especially pronounced for residues 58 and 61. Clearly, binding affinity of LLhP for the lacO1 DNA site is sensitive to long-range changes in the linker. This result also raises the possibility that mutations at positions 58 and 61 co-evolved with changes in the DNA-binding site. In addition, repression measured in the absence and presence of effector ligand shows that allosteric response increases for several LLhP variants with substitutions at positions 48 and 55. Thus, while side chain variation at these sites does not generally dictate the presence or absence of allostery, the nature of the amino acid can modulate the response to effector.