The cyclin and cystathionine β-synthase (CBS) domain magnesium transport mediators, CNNMs, are key players in maintaining the homeostasis of magnesium in different organs. The human family includes four members, whose impaired activity causes diseases such as Jalili Syndrome or Familial Hypomagnesemia, but is also linked to neuropathologic disorders, altered blood pressure, and infertility. Recent findings demonstrated that CNNMs are associated with the highly oncogenic phosphatases of the regenerating liver to promote tumor growth and metastasis, which has attracted renewed focus on their potential exploitation as targets for cancer treatment. However, the exact function of CNNMs remains unclear and is subject to debate, proposed as either direct transporters, sensors, or homeostatic factors. This review gathers the current structural knowledge on the CNNM family, highlighting similarities and differences with the closely related structural partners such as the bacterial Mg²⁺/Co²⁺ efflux protein CorC and the Mg²⁺ channel MgtE.

Cystathionine β-synthase (CBS) catalyzes the condensation of serine and homocysteine to water and cystathionine, which is then hydrolyzed to cysteine, α-ketobutyrate and ammonia by cystathionine γ-lyase (CGL) in the reverse transsulfuration pathway. The protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, includes both CBS and CGL enzymes. We have recently reported that the putative T. gondii CGL gene encodes a functional enzyme. Herein, we cloned and biochemically characterized cDNA encoding CBS from T. gondii (TgCBS), which represents a first example of protozoan CBS that does not bind heme but possesses two C-terminal CBS domains. We demonstrated that TgCBS can use both serine and O-acetylserine to produce cystathionine, converting these substrates to an aminoacrylate intermediate as part of a PLP-catalyzed β-replacement reaction. Besides a role in cysteine biosynthesis, TgCBS can also efficiently produce hydrogen sulfide, preferentially via condensation of cysteine and homocysteine. Unlike the human counterpart and similar to CBS enzymes from lower organisms, the TgCBS activity is not stimulated by S-adenosylmethionine. This study establishes the presence of an intact functional reverse transsulfuration pathway in T. gondii and demonstrates the crucial role of TgCBS in biogenesis of H₂S.

The four member family of “Cyclin and Cystathionine β-synthase (CBS) domain divalent metal cation transport mediators”, CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg²⁺-efflux capacity, thus resulting in an increased intracellular Mg²⁺ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg²⁺, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg²⁺ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.

In higher organisms, epithelia separate compartments in order to guarantee their proper function. Such structures are able to seal but also to allow substances to pass. Within the paracellular pathway, a supramolecular structure, the tight junction transport is largely controlled by the temporospatial regulation of its major protein family called claudins. Besides the fact that the expression of claudins has been identified in different forms of human diseases like cancer, clearly defined mutations in the corresponding claudin genes have been shown to cause distinct human disorders. Such disorders comprise the skin and its adjacent structures, liver, kidney, the inner ear, and the eye. From the phenotype analysis, it has also become clear that different claudins can cause a complex phenotype when expressed in different organs. To gain deeper insights into the physiology and pathophysiology of claudin-associated disorders, several mouse models have been generated. In order to model human disorders in detail, they have been designed either as full knockouts, knock-downs or knock-ins by a variety of techniques. Here, we review human disorders caused by CLDN mutations and their corresponding mouse models that have been generated thus far and assess their usefulness as a model for the corresponding human disorder.

Cystathionine beta-synthase (CBS) is a key regulator of homocysteine metabolism. Although eukaryotic CBS have a similar domain architecture with a catalytic core and a C-terminal Bateman module, their regulation varies widely across phyla. In human CBS (HsCBS), the C-terminus has an autoinhibitory effect by acting as a cap that avoids the entry of substrates into the catalytic site. The binding of the allosteric modulator AdoMet to this region alleviates this cap, allowing the protein to progress from a basal toward an activated state. The same activation is obtained by artificial removal or heat-denaturation of the Bateman module. Recently, we reported the crystal structure of CBS from Toxoplasma gondii (TgCBS) showing that the enzyme assembles into basket-like dimers similar to the basal conformers of HsCBS. These findings would suggest a similar lid function for the Bateman module which, as in HsCBS, should relax in the absence of the C-terminal module. However, herein we demonstrate that, in contrast with HsCBS, removal of the Bateman module in TgCBS through deletion mutagenesis, limited proteolysis, or thermal denaturation has no effects on its activity, oligomerization, and thermal stability. This opposite behavior we have now found in TgCBS provides evidence of a novel type of CBS regulation.

Cystathionine β-synthase (CBS), the pivotal enzyme of the reverse transsulfuration pathway, catalyzes the pyridoxal-5’-phosphate-dependent condensation of serine with homocysteine to form cystathionine. Additionally, CBS performs alternative reactions that use homocysteine and cysteine as substrates leading to the endogenous biosynthesis of hydrogen sulfide (H ₂ S), an important signal transducer in many physiological and pathological processes. Toxoplasma gondii, the causative agent of toxoplasmosis, encodes a functional CBS ( Tg CBS) that contrary to human CBS, is not allosterically regulated by S-adenosylmethionine and can use both, Ser and O -acetylserine (OAS) as substrates. Tg CBS is also strongly implicated in the production of H ₂ S, and thus involved in redox homeostasis of the parasite. Here, we report its crystal structure, the first CBS from a protozoan described so far. Our data reveals a basal-like fold that unexpectedly differs from the active conformations found in other organisms, but structurally similar to the pathogenic activated mutant D444N of the human enzyme.