Structural insight into the molecular mechanism of allosteric activation of human cystathionine β-synthase by S-adenosylmethionine

Cystathionine β-synthase (CBS) is a heme-dependent and pyridoxal-5′-phosphate–dependent protein that controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H₂S. Deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur amino acid metabolism. In contrast to CBSs from lower organisms, human CBS (hCBS) is allosterically activated by S-adenosylmethionine (AdoMet), which binds to the regulatory domain and triggers a conformational change that allows the protein to progress from the basal toward the activated state. The structural basis of the underlying molecular mechanism has remained elusive so far. Here, we present the structure of hCBS with bound AdoMet, revealing the activated conformation of the human enzyme. Binding of AdoMet triggers a conformational change in the Bateman module of the regulatory domain that favors its association with a Bateman module of the complementary subunit to form an antiparallel CBS module. Such an arrangement is very similar to that found in the constitutively activated insect CBS. In the presence of AdoMet, the autoinhibition exerted by the regulatory region is eliminated, allowing for improved access of substrates to the catalytic pocket. Based on the availability of both the basal and the activated structures, we discuss the mechanism of hCBS activation by AdoMet and the properties of the AdoMet binding site, as well as the responsiveness of the enzyme to its allosteric regulator. The structure described herein paves the way for the rational design of compounds modulating hCBS activity and thus transsulfuration, redox status, and H₂S biogenesis.Cystathionine β-synthase (CBS; EC 4.2.1.22) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of the hydroxyl group of l-serine (Ser) by l-homocysteine (Hcy), yielding cystathionine (Cth) (1). A deficient activity of human CBS (hCBS) is the cause of classical homocystinuria [CBS-deficient homocystinuria (CBSDH); Online Mendelian Inheritance in Man (OMIM) no. 236200], an autosomal, recessive inborn error of sulfur amino acid metabolism, characterized by increased levels of Hcy in plasma and urine. CBSDH manifests as a combination of connective tissue defects, skeletal deformities, vascular thrombosis, and mental retardation (2).The hCBS is a homotetrameric enzyme whose subunits are organized into three structural domains. The N-terminal region binds heme and is thought to function in redox sensing and/or enzyme folding (3, 4). The central catalytic core shows the fold of the type II family PLP-dependent enzymes (5, 6). Finally, the C-terminal region consists of a tandem pair of CBS motifs (7–9) that bind S-adenosylmethionine (AdoMet) and lead to an increase in catalytic activity by up to fivefold (10, 11). The CBS motif pair, commonly known as a “Bateman module” (12, 13), is responsible for CBS subunit tetramerization (14, 15). The presence of pathogenic missense mutations in this region often does not impair enzyme activity but typically interferes with binding of AdoMet and/or the enzyme’s activation by AdoMet (15–17). Removal of the regulatory region leads to a dimer with much increased activity (14, 15). Recently, we showed that removal of residues 516–525, forming a flexible loop of the CBS2 motif of hCBS, yields dimeric species (hCBSΔ516–525) with intact AdoMet binding capacity and activity responsiveness to AdoMet similar to a native hCBS WT (18).hCBS is regulated by a complex molecular mechanism that remains poorly understood. More than a decade ago, we and others hypothesized that hCBS might exist in two different conformations: a “basal” state with low activity, where the C-terminal regulatory domain would restrict the access of substrates into the catalytic site, and an AdoMet-bound “activated” state, where the AdoMet-induced conformational change would allow for enzyme activation (16, 19). Recently, we have unveiled the relative orientations of the regulatory and catalytic domains in hCBS (18), which were in a striking contrast to those of both the previous in silico models (20, 21) and the Drosophila melanogaster (dCBS) structure (22). Our data showed that, although the pairing mode and the orientation of catalytic cores are similar in both insect dCBS and hCBS, the position of their regulatory domains is markedly different (18). In the basal state, the Bateman modules from each hCBS unit are far apart and do not interact with each other, being placed just above the entrance of the catalytic site of the complementary subunit, thus hampering the access of substrates into this cavity. Our hCBSΔ516–525 structure additionally revealed the presence of two major cavities in the Bateman module, S1 and S2, one of which (S2) is solvent-exposed and probably represents the primary binding site for AdoMet (18). These findings are in agreement with the much higher basal activity of dCBS and its inability to bind or to be regulated by AdoMet (23, 24) and suggest that the structural basis underlying the regulation of the human enzyme markedly differs from CBS regulation in insects or yeast (24). Taken together, the available data indicate that binding of AdoMet to the Bateman module weakens the interaction between the regulatory domain and the catalytic core although the mechanism and the magnitude of the underlying structural effect are still under debate (16, 19, 25–27).To solve the molecular mechanism of hCBS regulation by AdoMet, we have analyzed the crystals of an engineered hCBSΔ516–525 protein that bears the mutation E201S, which potentially weakens and/or disrupts the interaction between the Bateman module and the catalytic core (Fig. 1A), thus favoring the activation of the enzyme. The data presented here fill a long-sought structural gap by unraveling the crystal structure of AdoMet-bound hCBS, thus providing the overall fold of the enzyme in its activated conformation and the identity of the AdoMet binding sites. Comparison with the structures of hCBS in basal conformation and constitutively activated dCBS was instrumental in the understanding of the regulatory role played by the C-terminal domain as well as the effect of some of the pathogenic mutations in the activation and/or inhibition of this key molecule of transsulfuration.Open in a separate windowFig. 1.Interactions between protein domains in basal hCBS. (A) In hCBSΔ516–525, residues Y484, N463, and S466 anchor the Bateman module (blue) to the protein core (gray) through H-bonds with the residues E201 and D198 from the loop L191–202, thus occluding the entrance to the catalytic pocket. (B) The CBS-specific activity of selected hCBS variants in the absence (blue bars) and the presence (red bars) of 300 µM AdoMet. hCBS enzyme species marked with “Δ” lack residues 516–525 and form dimers.     

Intramuscular epithelioid hemangioma of the parapharyngeal space

A clinical case of epithelioid hemangioma of the scalene muscle that occupied the parapharyngeal space is reported. The patient was a 34 year old man with a 2-month history of sensation of pharyngeal foreign body and mild dysphagia. The exploration revealed a tumor of the posterior and lateral wall of the oropharynx that extended from the rhinopharynx to the hypopharynx. The diagnostic sequence included CT, MRI, Doppler echography, and arteriography, which identified a right post-styloid tumor located behind and medial to the jugular vein, internal carotid artery, and vagal nerve, but did not affect arterial blood flow. The patient underwent surgical treatment consisting of lateral cervicotomy, tumor excision, and histological study.     

Subgingival Epstein-Barr and cytomegalovirus occurrence in pregnancy gingivitis

Background: Although recent studies focused on the role of human herpesviruses in various types of periodontal disease, there was a lack of information in these reports regarding the role of pregnancy gingivitis. The aim of this study is to determine the correlation between pregnancy and the subgingival virus presence and their relationship with clinical parameters. Methods: Seventy pregnant and 40 non‐pregnant women were examined for gingival and plaque indices, bleeding on probing (BOP), and clinical probing depths (PDs) from the whole dentition. Subgingival plaque samples were obtained from sites showing signs of gingivitis and healthy sites. The polymerase chain reaction methodology was used to detect cytomegalovirus (CMV) and Epstein‐Barr virus (EBV) from plaque samples. Results: Our results show that gingivitis lesions in 27 (38.6%) and 10 (14.3%) pregnant patients were positive for EBV and CMV, respectively. In the non‐pregnant group, EBV and CMV were detected in six (15%) and eight (20%) lesions, respectively. A statistically significant difference (P <0.01) was found between the subgingival occurrence of EBV in the two groups. In gingivitis sites, clinical PDs were affected by gestation (P <0.001) and the occurrence of EBV (P <0.001). In healthy sites, clinical PDs were affected by gestation (P <0.05), and BOP was affected by the occurrence of CMV and EBV (P <0.001). Conclusion: Our data indicate that pregnancy increased the risk of the presence of subgingival EBV in pregnant women by 3.647 times more than in non‐pregnant women.     

Focal xanthogranulomatous pyelonephritis simulating malignancy in children

Xantogranulomatous pyelonephritis is a severe chronic form of renal parenquimal infection that usually results in diffuse renal destruction. An unusual case of xantho-granulomatous pyelonephritis in a child is reported which presented as a focal mass without calculus in a functioning kidney and was diagnosed as a renal tumor.     

Mandibular Metastasis of a Signet Ring Cell Carcinoma of the Breast in a Patient who Underwent Bilateral Mastectomy more than 25 Years Earlier

Summary

Background

Metastatic tumors account for less than 1% of all malignant tumors occurring in the oral cavity.

Case Report

The clinical case of a 94-year-old patient with a mandibular tumor is reported here. The patient had undergone bilateral mastectomy more than 25 years before. An immunohistochemical study found hormone receptors in signet ring cells, suggesting a diagnosis of breast cancer metastasis.

Conclusion

Immunohistochemical diagnosis and antineoplastic hormone therapy is the cornerstone in the management of this clinical case.Key Words: Mandible, Metastasis, Breast cancer, Signet ring cells     

Midfacial granuloma syndrome. A clinic and pathological report on four patients

Four cases of midfacial necrotizing lesions are reported. All patients were males with ages ranging from 25 to 76 years. The relationship between subjective symptoms and laboratory data prior to therapy (leukopenia, elevated ESR, increment of IgA and IgG), as well as between fever crisis with sweats and chills and the progression of the lesions were pathognomonic clinical signs for us. In all cases, paranasal sinus and nasopharynx were involved. Middle ear, eye and kidney involvement was present in 2 cases, and joints lesions only in one. Three patients died (2 of sepsis and one from hemorrhage) despite therapy. A pleomorphic cellular infiltrate with atypical lymphocytes and a tendency to angiocentricity was found in these cases. Such features and PAP positivity to beta and kappa chains led us to consider these lesions as an extranodal B-lymphocyte lymphoma-like. In the fourth case the histological picture was that of a necrotizing granuloma with clustered giant cells. This patient, treated only with prednisone, had a total remission of his symptomatology up to 11 years after the onset of the disease.     

Squamous cell carcinoma of the male urethra mimicking a paravertebral abscess.

In the context of a misleading diagnosis of paraurethral abscess, we report a case of unrecognized metastatic squamous cell carcinoma of the male urethra that mimicked a picture of paravertebral abscess with accompanying massive caval vein thrombosis. Fine needle aspiration cytology of both paravertebral abscess and urethral nodule allowed the diagnosis. This report calls the attention of clinicians to think of a malignant process in case of a paravertebral abscess. Fine needle aspiration cytology once more proves a useful tool in the diagnosis of unsuspected malignancies.