This entry represents Midline-1. Midline-1 is associated with microtubules throughout the cell cycle, co-localising with cytoplasmic fibres in interphase and with the mitotic spindle and midbodies during mitosis and cytokinesis []. It has E3 ubiquitin ligase activity towards IGBP1, promoting its monoubiquitination, which results in deprotection of the catalytic subunit of protein phosphatase PP2A, and its subsequent degradation by polyubiquitination [, , ]. Defects in the midline-1 gene are the cause of Opitz GBBB syndrome 1 (OGS1), which is characterised by hypertelorism, genital-urinary defects, lip-palate-laryngotracheal clefts, developmental delay and congenital heart defects [, , ].
This entry represents the COS domain found in the C-I subfamily of the tripartite motif (TRIM) family of E3 ubiquitin ligases.The C-1 subfamily includes MID1 (TRIM18), MID2 (TRIM1), TRIM9, TNL (TRIM67), TRIM36 and TRIFIC (TRIM46) [, , ]. The C-I subfamily contains C-terminal FNIII and B30.2 domains which are associated with microtubules [, ]. C-I subfamily members also contain a relatively conserved region of approximately 60 amino acids, termed the C-terminal subgroup One Signature (COS) domain []. The human Midline-1 (MID1) COS domain has a helix-loop-helix structure in which the N- and C-terminal ends are in close proximity []. Deletion of the human MID1 COS domain does not affect MID1 dimerisation but disrupts localisation to the microtubules [].The COS domain is also observed within the TRIM C-II subgroup, which consists of the MURF1-3 proteins that do not contain the FNIII and B30.2domains. MUF1-3 proteins are also associated with microtubules [].
In Saccharomyces cerevisiae, Mid1 is a yeast plasma membrane protein required for Ca2+ influx induced by the mating pheromone alpha-factor during the mating process [, , ]. The protein is composed of 548-amino-acid residues, contains four hydrophobic regions (H1, H2, H3 and H4) and two cysteine-rich regions (C1 and C2) at the C terminus. H1 appears to be a signal sequence necessary for the alpha-factor-induced delivery to the plasma membrane. The region from H1 to H3 is required for the localisation of Mid1 in the plasma and ER membranes. C1 and C2 are thought to be involved in oligomerisation via the formation of disulphide bonds. Trafficking of Mid1-GFP to the plasma membrane is dependent on the N-glycosylation of Mid1 and the transporter protein Sec12. This suggests that the trafficking of Mid1-GFP to the plasma membrane requires a Sec12-dependent pathway from the ER to the Golgi, and that Mid1 is recruited via a Sec6- and Sec7-independent pathway from the Golgi to the plasma membrane.This entry also includes Ehs1 from Schizosaccharomyces pombe. Ehs1 is required for Ca2+ influx and for vitality of cells in a late, pheromone-induced event of the mating process requiring calcium-induced signaling [].
MID2 shares high structural and functional similarities with MID1, an ubiquitin E3 ligase that regulates the turnover of microtubular protein phosphatase 2Ac. MID2 interacts with Astrin (a microtubule-organizing protein) and regulates Astrin levels to promote cell division []. MID1 and MID2 may play roles in regulating tissue remodelling in early development [].Mutations in the MID2 gene cause mental retardation, X-linked 101 (MRX101) [].
This entry represents B-box-type zinc finger domains, which are around 40 residues in length. B-box zinc fingers can be divided into two groups, where types 1 and 2 B-box domains differ in their consensus sequence and in the spacing of the 7-8 zinc-binding residues. Several proteins contain both types 1 and 2 B-boxes, suggesting some level of cooperativity between these two domains. B-box domains are found in over 1500 proteins from a variety of organisms. They are found in TRIM (tripartite motif) proteins that consist of an N-terminal RING finger (originally called an A-box), followed by 1-2 B-box domains and a coiled-coil domain (also called RBCC for Ring, B-box, Coiled-Coil). TRIM proteins contain a type 2 B-box domain, and may also contain a type 1 B-box. In proteins that do not contain RING or coiled-coil domains, the B-box domain is primarily type 2. Many type 2 B-box proteins are involved in ubiquitination. Proteins containing a B-box zinc finger domain include transcription factors, ribonucleoproteins and proto-oncoproteins; for example, MID1, MID2, TRIM9, TNL, TRIM36, TRIM63, TRIFIC, NCL1 and CONSTANS-like proteins [].The microtubule-associated E3 ligase MID1 () contains a type 1 B-box zinc finger domain. MID1 specifically binds Alpha-4, which in turn recruits the catalytic subunit of phosphatase 2A (PP2Ac). This complex is required for targeting of PP2Ac for proteasome-mediated degradation. The MID1 B-box coordinates two zinc ions and adopts a β/β/α cross-brace structure similar to that of ZZ, PHD, RING and FYVE zinc fingers [, ].
This domain, consisting of the distinct N-terminal PRY subdomain followed by the SPRY subdomain, is found at the C terminus of TRIM1 (also known as MID2 or midline 2). MID2 and its close homologue, TRIM18 (also known as MID1), both contain a B30.2-like domain at their C terminus and a single fibronectin type III (FN3) motif between it and their N-terminal RBCC domain. Mid1 and Mid2 are ubiquitin ligases that regulate microtubule dynamics and whose mutation is associated with X-linked developmental disorders. Mid2 regulates cell division through the ubiquitination of astrin on K409, which is critical for its degradation and proper cytokinesis [].
Zinc finger (Znf) domains are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not; instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise the finger-like folds. They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein and/or lipid substrates [, , , , ]. Their binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. There are many superfamilies of Znf motifs, varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g. some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organisation, epithelial development, cell adhesion, protein folding, chromatin remodelling and zinc sensing, to name but a few []. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. This entry represents B-box-type zinc finger domains, which are around 40 residues in length. B-box zinc fingers can be divided into two groups, where types 1 and 2 B-box domains differ in their consensus sequence and in the spacing of the 7-8 zinc-binding residues. Several proteins contain both types 1 and 2 B-boxes, suggesting some level of cooperativity between these two domains. B-box domains are found in over 1500 proteins from a variety of organisms. They are found in TRIM (tripartite motif) proteins that consist of an N-terminal RING finger (originally called an A-box), followed by 1-2 B-box domains and a coiled-coil domain (also called RBCC for Ring, B-box, Coiled-Coil). TRIM proteins contain a type 2 B-box domain, and may also contain a type 1 B-box. In proteins that do not contain RING or coiled-coil domains, the B-box domain is primarily type 2. Many type 2 B-box proteins are involved in ubiquitination. Proteins containing a B-box zinc finger domain include transcription factors, ribonucleoproteins and proto-oncoproteins; for example, MID1, MID2, TRIM9, TNL, TRIM36, TRIM63, TRIFIC, NCL1 and CONSTANS-like proteins [].The microtubule-associated E3 ligase MID1 () contains a type 1 B-box zinc finger domain. MID1 specifically binds Alpha-4, which in turn recruits the catalytic subunit of phosphatase 2A (PP2Ac). This complex is required for targeting of PP2Ac for proteasome-mediated degradation. The MID1 B-box coordinates two zinc ions and adopts a β/β/α cross-brace structure similar to that of ZZ, PHD, RING and FYVE zinc fingers [, ].
N-terminal RING finger/B-box/coiled coil (RBCC) or tripartite motif (TRIM) proteins, which are found in metazoa, are involved in a vast array of intracellular functions. They appear to function as part of large protein complexes and possess ubiquitin-protein isopeptide ligase activity. The following RBCC proteins contain an ~60-residue COS (C-terminal subgroup one signature) domain, which is also found in a distantly related non-RBCC microtubule-binding protein, GLFND:Vertebrate MID1 and MID2, which associate with microtubules through homo- and heterodimerizationAnimal TRIM9, which plays a regulatory role in synaptic vesicle exocytosisMammalian TRIM nine-like (TNL)Mammalian TRIM36, which could play a regulatory role in exocytosis of the sperm vesicleMammalian tripartite, fibronectin type III and C-terminal B30.2/SPRY (TRIFIC)Mammalian muscle-specific RING finger (MURF) family. MURF proteins have an ability to form both homo- and heterodimers with each other and to associate with the microtubule cytoskeletonIn addition to RBCC, the COS domain is also found in association with B30.2/SPRY or fibronectin type-III (FN3) domains.The COS domain is predicted to consist of two α-helical coils [].
