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Ðîëü ñâåðõñåìåéñòâà êàäãåðèíîâ

Thinking outside the cell: how cadherins drive adhesion
Julia Brasch, Oliver J. Harrison, Barry Honig, Lawrence Shapiro
Trends in Cell. Biol. Volume 22, Issue 6, June 2012, Pages 299–310 http://dx.doi.org/10.1016/j.tcb.2012.03.004

Cadherins are a superfamily of cell surface glycoproteins whose ectodomains contain multiple repeats of β-sandwich extracellular cadherin (EC) domains that adopt a similar fold to immunoglobulin domains. The best characterized cadherins are the vertebrate ‘classical’ cadherins, which mediate adhesion via trans homodimerization between their membrane-distal EC1 domains that extend from apposed cells, and assemble intercellular adherens junctions through cis clustering. To form mature trans adhesive dimers, cadherin domains from apposed cells dimerize in a ‘strand-swapped’ conformation. This occurs in a two-step binding process involving a fast-binding intermediate called the ‘X-dimer’. Trans dimers are less flexible than cadherin monomers, a factor that drives junction assembly following cell–cell contact by reducing the entropic cost associated with the formation of lateral cis oligomers. Cadherins outside the classical subfamily appear to have evolved distinct adhesive mechanisms that are only now beginning to be understood.


Ðèñóíêè ê ñòàòüå



The classical cadherin family


Êàäãåðèíû ñîñòàâëÿþò êðóïíîå ñâåðõñåìåéñòâî ðåöåïòîðîâ êëåòî÷íîé ïîâåðõíîñòè, ìíîãèå èç êîòîðûõ ó÷àñòâóþò â çàâèñèìîì îò êàëüöèÿ ìåæêëåòî÷íîì ðàñïîçíàâàíèè è àäãåçèè. Êàäãåðèíû îáíàðóæèâàþòñÿ ó øèðîêîãî êðóãà âèäîâ îò îäíîêëåòî÷íûõ îðãàíèçìîâ ñ ìíîãîêëåòî÷íûìè æèçíåííûìè ñòàäèÿìè1,2 äî ìëåêîïèòàþùèõ, ó êîòîðûõ îíè ó÷àñòâóþò â ìîðôîãåíåòè÷åñêèõ ïðîöåññàõ, òàêèõ êàê ðàçäåëåíèå ýìáðèîíàëüíûõ êëåòî÷íûõ ñëî¸â, ôîðìèðîâàíèå ñèíàïñîâ è ñïåöèôè÷íîñòè â ÖÍÑ3,4, ìåõàíîòðàíñäóêöèè5,6, ïåðåäà÷å êëåòî÷íûõ ñèãíàëîâ7,8, è ôèçè÷åñêîì ãîìåîñòàçå çðåëûõ òêàíåé9,10.  ñîîòâåòñòâèè ñ èõ ðîëÿìè, ñíèæåíèå ýêñïðåññèè êàäãåðèíîâ ìîæåò ïîçâîëèòü êëåòêàì èçáåæàòü íîðìàëüíûõ ïîòðåáíîñòåé â êëåòî÷íîé àäãåçèè äëÿ æèçíåñïîñîáíîñòè11-13, ýòî ÿâëÿåòñÿ îáùèì ïðèçíàêîì ìåòàñòàçèðîâàíèÿ.
×ëåíû ñâåðõåìåéñòâà cadherin îïðåäåëÿþòñÿ ïî îáùåìó ñòðóêòóðíîìó êîìïîíåíòó, EC äîìåíe - ïðèáëèçèòåëüíî â 110 îñòàòêîâ β-fold äîìåíó - è cadherins ìîãóò áûòü êëàññèôèöèðîâàíû íà íåñêîëüêî ïîäñåìåéñòâ, áàçèðóÿñü íà êîëè÷åñòâå è ðàñïîëîæåíèè EC äîìåíîâ (Box 1, Figure I). Äëÿ ëó÷øåãî ïîíèìàíèÿ ýòèõ ïîäñåìåéñòâ êëàññè÷åñêèå êàäãåðèíû ïîçâîíî÷íûõ ïðåäñòàâëåíû 6 'type I' è 13 'type II' cadherins â òèïè÷íûõ ãåíîìàõ ïîçâîíî÷íûõ, êîòîðûå îáëàäàþò çàêîíñåðâèðîâàííûì öèòîïëàçìàòè÷åñêèì äîìåíîì è ýêòîäîìåíîì, ñîäåðæàùèì 5 òàíäåìíûõ EC äîìåíîâ (Figure 1). Ëèíêåðû ìåæäó ïîñëåäîâàòåëüíûìè EC äîìåíàìè ñòàáèëèçèðóþòñÿ êàæäûé ñ ïîìîùüþ ñâÿçûâàíèÿ òðåõ èîíîâ Ca2+, ïðèâîäÿùèõ â ðåçóëüòàòå ê õàðàêòåðíîìó èçãèáó ýêòîäåìåíà (Figure 1). Ðîëü ñâÿçûâàíèÿ Ca2+ â êëàññè÷åñêèõ êàäãåðèíàõ îáåñïå÷èâàåò àäãåçèþ.



Box 1. Meeting the family Cadherins are membrane associated glycoproteins, many of which function in calcium-dependent cell adhesion or recognition processes. Each EC domain comprises a seven stranded ß-barrel 23, 24, 25, 26, 30, 51, 65, 70, 71, 75 and 80 with the N and C termini located on opposite sides allowing consecutive domains to be arranged in tandem. Most EC domains contain conserved Ca2+-binding sites that coordinate three Ca2+ ions in the linker regions between consecutive domains [24], rigidifying the ectodomain structure [81] and protecting it from proteolysis [82] (Box 2). Less frequently, and mostly in very long cadherins, canonical EC domains can lack Ca2+-binding residues resulting in Ca2+-free linker regions, suggesting flexibility that could result in more globular overall structures 2 and 75. The number of EC domains, overall domain organization and other sequence characteristics vary widely between different cadherins, dividing the superfamily into several subfamilies (Figure I) 83 and 84. Vertebrate classical type I and type II cadherins are single-pass transmembrane proteins with ectodomains comprising five EC repeats (after removal of the N-terminal prodomain), and a short, highly conserved cytoplasmic domain with binding motifs for the armadillo proteins p120 and ß-catenin (reviewed in [8]). Desmosomal cadherins, expressed in all vertebrate animals, have a domain organization similar to that of classical cadherins (reviewed in [60]). However, they are attached via distinct cytoplasmic proteins to intermediate filaments forming specialized cell-cell junctions, referred to as desmosomes, in tissues exposed to high mechanical stress. The largest cadherin subfamily is the protocadherins, divided into the gene-clustered a-, ß- and ?- and non-clustered protocadherins (reviewed in 85 and 86, respectively). They are single-pass transmembrane proteins with six or seven EC domains and distinct cytoplasmic domains, and are expressed primarily in the nervous system of mammals. Clustered protocadherins are thought to play an important role in neural patterning 66 and 85. Other subfamilies are more divergent; for example, Flamingo/CELSR cadherins, which mediate planar cell polarity in vertebrates and invertebrates, have nine EC repeats, EGF, laminin-G like and hormone receptor-like domains and, uniquely in the cadherin family, a seven-pass transmembrane structure [87]. Some atypical cadherins, such as FAT and Dachsous, are involved in adhesion-mediated signaling and planar cell polarity [88]. These cadherins have many EC domains, but have a close phylogenetic sequence resemblance to protocadherins in their N-terminal region [2]. Invertebrate 'classical' cadherins, typified by Drosophila N- and E-cadherin, are found in adherens junction-like structures but deviate greatly in their domain organization. The heterophilic adhesive pair cadherin-23 and protocadherin-15 appear to form a long braided structure, the 'tip-link', which is involved in auditory mechanotransduction 6 and 89. Each vertebrate genome contains a solitary truncated (T-) cadherin [90], which regulates neurite outgrowth and has a similar overall domain organization to the ectodomain of classical cadherins, but the transmembrane and cytoplasmic domain are replaced by a GPI anchor. T-cadherin binds adhesively through the 'X-dimer' interface [51], which functions as a binding intermediate in vertebrate classical cadherins [48].




Figure I. Schematic representation of members of the cadherin family, which share a common structural motif: the EC domain. (a) Typical folding of an EC domain shown in ribbon representation (top panel from pdb-ID: 1L3W). Seven antiparallel ß-strands (A-G) assemble two ß-sheets as shown in the topology diagram (lower panel). Note that the A strand is split into two halves, the A* and A strands. These are connected by a loop, referred to as the 'hinge'. Three Ca2+ ions (green spheres) are coordinated between consecutive EC domains. (b) Schematic representation of overall domain organization of selected cadherin family members. All cadherins have two or more EC domains in their extracellular regions (blue ovals, numbered from membrane distal to membrane proximal domain), which can also contain non-EC domains such as EGF-repeats (green rectangles), laminin A G domains (cyan diamonds) and flamingo boxes (pink oval). Some cadherins have, in addition to the signal peptide, a prodomain (grey ovals) that is removed by a furin protease on the cell surface. Hashed domains indicate four or more EC domains omitted for clarity. *The first EC domain of Drosophila E- (DE) and N- (DN) cadherin is predicted from sequence analysis [75].



Figure 1. Overall architecture of classical cadherins. The extracellular domain of C-cadherin (pdb-ID: 1L3W) is depicted as a ribbon diagram (orange). Ca2+ ions (green spheres) are coordinated between consecutive domains, stabilizing an overall curved shape of the ectodomain, with an angle of close to 90° between domains EC1 and EC5. The structure of the stalk region, the transmembrane domain and parts of the intracellular domain are unknown and are shown as dotted lines. The cytoplasmic domain of cadherins binds to intracellular binding partners p120 (green barrels representing a-helices; pdb-ID: 3L6X) in the juxta-membrane region and ß-catenin (blue barrels representing a-helices; pdb-ID: 1I7X) in the C-terminal region. ß-catenin interacts with a-catenin, which in turn binds to actin filaments linking cadherins to the cytoskeleton. The depicted orientation, position and size of the intracellular binding partners relative to each other and to C-cadherin are schematic; the overall structural arrangement of the cytoplasmic side of adherens junctions is unknown.

Box 2. Calcium dependence of cadherin adhesion Cadherins are named for the dependence of their adhesive function on the presence of extracellular calcium. Before their structures were known, it was speculated that Ca2+ ions might bridge the adhesive interface. However, the role of calcium in cadherin function is far more complex. Calcium binds to cadherins at stereotyped binding sites situated between successive EC domains. Each of these sites binds three Ca2+ ions in a highly cooperative manner such that each five-domain classical cadherin coordinates twelve Ca2+ ions in total 17, 24 and 80. The binding affinities of the Ca2+ sites vary, but all bind with a dissociation constant (KD) lower than the Ca2+ concentration characteristic of the extracellular milieu, approximately 1 mM 91 and 92. Thus, it is expected that cadherin ectodomains will be fully Ca2+-occupied under physiological conditions.
Ñåãîäíÿ èçâåñòíû òðè ðîëè äëÿ ñâÿçûâàíèÿ Ca2+ â êëàññè÷åñêèõ êàäãåðèíàõ. Ïåðâàÿ ýòî óñèëåíèå æåñòêîñòè ýêòîäåìåíà òàê, ÷òî îí ïðèíèìàåò õàðàêòåðíóþ ôîðìó ïîëóìåñÿöà ectodomain so that it adopts a characteristic crescent shape [81], although this structure retains considerable conformational flexibility 55 and 70. The crescent shape is critical to adhesive binding because the axes of the membrane-distal and membrane-proximal EC domains must be approximately 90° apart to satisfy the geometrical requirements of trans binding 17 and 24. Notably, chelation of Ca2+ leads to the loss of trans binding and its concomitant replacement by binding to other cadherins on the same cell through the adhesive interface [93]. Thus, Ca2+-mediated rigidification is critical to adhesive trans binding.
A second role for Ca2+ ions is in defining the structure of the X-dimer interface surfaces. The X-dimer binding intermediate of classical cadherins is centered around the EC1-EC2 Ca2+ binding region, which is unstructured in the absence of Ca2+48, 51, 80, 94 and 95. Thus, in the absence of Ca2+, the mature adhesive strand-swap interface is likely to be kinetically unfavorable due to the slow exchange inherent in domain swap binding.
The third role for Ca2+ involves direct energetic effects on strand swapping. NMR experiments [46] and molecular simulations [96] reveal that Ca2+ ligation favors the opening of the A strand. The underlying molecular mechanism has recently been described [34] and is discussed in the text.


Êëàññè÷åñêèå êàäãåðèíû ïðåäñòàâëÿþò ñîáîé ïðîòîòèïè÷åñêèé ïðèìåð êàëüöèé-çàâèñèìîé ãîìîôèëüíîé ìåæêëåòî÷íîé àäãåçèè. Îíè ÷àñòî êîíöåíòðèðóþòñÿ â ñëèï÷èâûõ ñîåäèíåíèÿõ (rev. [14]), â ñïåöèàëèçèðîâàííûõ ìåæêëåòî÷íûõ ñëèï÷èâûõ ñòðóêòóðàõ, õàðàêòåðèçóþùèõñÿ ïàðàëëåëüíûì ñìûêàíèåì ïëàçìàòè÷åñêèõ ìåìáðàí ñ ïðîñòðàíñòâîì ìåæäó ìåìáðàíàìè ïðèáëèçèòåëüíî â 15-30 nm.  ýòèõ ñîåäèíåíèÿõ êàäãåðèíû îáðàçóþò ïîïåðå÷íûå ìîñòèêè, ñâÿçûâàþùèå ïðîñòðàíñòâî ìåæäó ìåìáðàíàìè ïîñðåäñòâîì ñâîèõ ýêòîäîìåíîâ, òîãäà êàê èõ öèòîïëàçìàòè÷åñêèå äîìåíû ñâÿçàíû ñ àäàïòîðíûìè áåëêàìè β-catenin, êîòîðûé ñâÿçûâàåò êàäãåðèíû êîñâåííî ñ öèòîñêåëåòîì (rev. [8]), è p120 catenin, êîòîðûé ðåãóëèðóåò îáîðîò êàäãåðèíà è ìîäóëèðóåò ñáîðêó àêòèíà (rev. 15 and 16).
Íåäàâíèå èññëåäîâàíèÿ ïîäòâåðäèëè, ÷òî ýêòîäîìåíû êëàññè÷åñêèõ êàäãåðèíîâ, â îòñóòñòâèå öèòîïëàçìàòè÷åñêèõ ðåãèîíîâ, äîñòàòî÷íû äëÿ óïðàâëåíèÿ èíèöèàëüíîé ñáîðêîé ñëèï÷èâûõ ñîåäèíåíèé17-19. Ýòîò ïðîöåññ îáåñïå÷èâàåòñÿ êîîïåðàòèâíûì îáðàçîâàíèåì ðàçíîîáðàçíûõ cadherin-cadherin èíòåðôåéñîâ â öèñ (íà îäíîé è òîé æå êëåòêå) è â òðàíñ ïîëîæåíèè (íà ðàçíûõ êëåòêàõ).

Extracellular cadherin domains drive adhesion from outside the cell


Îòíîñèòåëüíûå âêëàäû â àäãåçèþ âíåêëåòî÷íîãî è âíóòðèêëåòî÷íîãî ðåãèîíîâ êëàññè÷åñêèõ êàäãåðèíîâ òîëüêî ïðîÿñíÿþòñÿ.  ðàííèõ èññëåäîâàíèÿõ èñïîëüçîâàëè êàäãåðèíû, èñêóññòâåííî ëèøåííûå ñàéòîâ ñâÿçûâàíèÿ p120 è β-catenin â öèòîïëàçìàòè÷åñêîì äîìåíå, äåìîíñòðèðóÿ ïîòåðþ àäãåçèè, ïîýòîìó ïåðâîíà÷àëüíî áûëî ñäåëàíî çàêëþ÷åíèå, ÷òî öèòîïëàçìàòè÷åñêèé àïïàðàò âàæåí äëÿ îáðàçîâàíèÿ êëàñòåðîâ êàäãåðèíîâ è ôîðìèðîâàíèÿ ñîåäèíåíèé [20]. Îäíàêî íåäàâíåå èññëåäîâàíèå ñ èñïîëüçîâàíèåì E-cadherin, ñõîäíûì îáðàçîì ëèøåííîãî ñàéòîâ ñâÿçûâàíèÿ β-catenin è p120, ÷òî âàæíî, ñâÿçûâàþùèé ìîòèâ äëÿ ýíäîöèòîòè÷åñêîãî clathrin àäàïòîðà áûë òàêæå äåëåòèðîâàí, âûÿâèëî ýôôåêòèâíîå îáðàçîâàíèå ñîåäèíåíèé17,18.  A431 êëåòêàõ, êîòîðûå áûëè ôîíîì ýêñïðåññèè äèêîãî òèïà E-cadherin, ýòè 'ëèøåííûå õâîñòà' cadherins ýôôåêòèâíî ðåêðóòèðîâàëèñü â ñëèï÷èâûå ñîåäèíåíèÿ äèêîãî òèïà [18]. Êðîìå òîãî, ó cadherin-äåôèöèòíûõ A431-D êëåòîê, ëèøåííûå õâîñòà êàäãåðèíû ôîðìèðîâàëè êëàñòåðû â ìåñòàõ êëåòî÷íûõ êîíòàêòîâ, êîòîðûå ñèëüíî íàïîìèíàëè ñëèï÷èâûå ñîåäèíåíèÿ äèêîãî òèïà [18]. Ñõîäíûì îáðàçîì, â ëåòêàõ MDCK II è äð. ýïèòåëèàëüíûõ êëåòî÷íûõ ëèíèÿõ, òðàíñôèöèðîâàííûå íå ñâÿçàííûå ñ catenin E-, N- è VE-cadherins [19] òàêæå ôîðìèðîâàëè ïîõîæèå íà ñëèï÷èâûå ñîåäèíåíèÿ êëàñòåðû íà áîêîâûõ ìåìáðàíàõ. Ýòè ðåçóëüòàòû ïîäòâåðäèëè, ÷òî öèòîïëàçìàòè÷åñêèé ðåãèîí íå ñóùåñòâåíåí äëÿ èíèöèàëüíîé ñáîðêè ñëèï÷èâûõ ñîåäèíåíèé, âñ¸ æå, ÷òîáû âèçóàëèçîâàòü ýòè ñîåäèíåíèÿ â êëåòêàõ íåîáõîäèìî óñòðàíåíèå ýíäîöèòîçà, ÷òîáû cadherin íà êëåòî÷íîé ïîâåðõíîñòè îñòàâàëñÿ äîëüøå17,18.
×òîáû ïðîâåðèòü ãèïîòåçó, ÷òî âíåêëåòî÷íûå äîìåíû êëàññè÷åñêèõ êàäãåðèíîâ ìîãóò ñîáèðàòüñÿ ñàìè, ÷òîáû ñôîðìèðîâàòü ïîäîáíûå ñîåäèíåíèÿì ñòðóêòóðû, äâå ãðóïïû îòäåëüíî ðàçðàáîòàëè áåñêëåòî÷íûå ëèïîñîìíûå ñèñòåìû, â êîòîðûõ ýêòîäîìåíû êàäãåðèíîâ, ñâÿçàííûå ñ ïîâåðõíîñòüþ ëèïîñîì, îöåíèâàëè â îòíîøåíèè àäãåçèè è îáðàçîâàíèÿ ñîåäèíåíèé17,21. Cryo-electron microscopy (cryo-EM) ïîêàçàëà, ÷òî êàê ïðèêðåïëåííûå ê ëèïîñîìàì E-cadherin [17] òàê è VE-cadherin [21] âíåêëåòî÷íûå äîìåíû ñîáèðàëèñü â êëàñòåðû â ìåñòàõ ñëèï÷èâûõ êîíòàêòîâ ìåæäó ëèïîñîìàìè è ôîðìèðîâàëè 'èñêóññòâåííûå ñëèï÷èâûå ñîåäèíåíèÿ', õàðàêòåðèçóþùèåñÿ îáðàçîâàíèåì ïëîòíûõ êëàñòåðîâ êàäãåðèíîâ è óïëîùåíèåì ñîïðèêàñàþùèõñÿ ìåìáðàí [17]. Èòàê, ýòè èññëåäîâàíèÿ ïðîäåìîíñòðèðîâàëè, ÷òî âíåêëåòî÷íûå äîìåíû êëàññè÷åñêèõ êàäãåðèíîâ ïîçâîíî÷íûõ äîñòàòî÷íû, ÷òîáû ñôîðìèðîâàòü ïåðâîíà÷àëüíûå ìåæêëåòî÷íûå êîíòàêòû ìîãóò ñîáèðàòü ïîõîæèå íà ñëèï÷èâûå ñîåäèíåíèÿ ñòðóêòóðû áåç ó÷àñòèÿ âêëàäà öèòîïëàçìàòè÷åñêîãî àïïàðàòà. Öèòîïëàçìàòè÷åñêèå âçàèìîäåéñòâèÿ ïðè ôîðìèðîâàíèè ñëèï÷èâûõ ñîåäèíåíèé, òàêèå êàê ñòàáèëèçàöèÿ ñîåäèíåíèé ïîñðåäñòâîì ðåêðóòèðîâàíèÿ àêòèíîâûõ âîëîêîí (rev. 14,22) , ñêîðåå âñåãî, ôóíêöèîíèðóþò ïîñëå ýòèõ èíèöèàëüíûõ âíåêëåòî÷íûõ ñîáûòèé. i

Mechanism of adhesive binding between single cadherin molecules from apposed cells


Ýêòîäîìåíû êëàññè÷åñêèõ êàäãåðèíîâ âûïÿ÷èâàþòñÿ ñî ñòîðîí ïðîòèâîïîëîæíûõ êëåòîê è îáðàçóþò òðàíñ àäãåçèâíûå ãîìîäèìåðû ïîñðåäñòâîì ñâîèõ äèñòàëüíûõ ìåìáðàííûõ EC1 äîìåíîâ, ñâÿçûâàþùèõ ìîñòèêàìè ïðîñòðàíñòâî ìåæäó ìåìáðàíàìè ñîñåäíèõ êëåòîê (Figure 2a). Èíòåðôåéñ, ëåæàùèé â îñíîâå ýòîãî âçàèìîäåéñòâèÿ, áûë îõàðàêòåðèçîâàí â äåòàëÿõ ñ àòîìíûì ðàçðåøåíèåì ñòðóêòóðû17, 23-27, ïîêàçûâàÿ. ÷òî âñå êëàññè÷åñêèå êàäãåðèíû îáëàäàþò îáùèì ìåõàíèçìîì ñâÿçûâàíèÿ, ïðè êîòîðîì íàèáîëåå N-òåðìèíàëüíàÿ ïîðöèÿ β-A íèòè, A* íèòè, îáìåíèâàþòñÿ ìåæäó EC1 äîìåíàìè ïðîòîìåðîâ àäãåçèâíûõ ïàðòíåðîâ (Figure 2a). Êëþ÷îì ýòîãî ìåõàíèçìà ÿâëÿåòñÿ ñòûêîâêà êîíñåðâàòèâíûõ ãèäðîôîáíûõ ÿêîðíûõ îñòàòêîâ, ðàñïîëîæåííûõ íà A* íèòè - òðèïòîôàíà â ïîçèöèè 2 (Trp2) äëÿ òèïà I êàäãåðèíîâ è Trp2 è Trp4 äëÿ òèïà II cadherins - ñ êîíñåðâàòèâíûì ãèäðîôîáíûì êàðìàíîì â òåëå EC1 äîìåíà ïàðòíåðà. Ôèçèîëîãè÷åñêîå çíà÷åíèå ýòîãî 'strand-swapped' àäãåçèâíîãî èíòåðôåéñà ïîäòâåðæäåíî ìíîãî÷èñëåííûìè ìóòàöèÿìè, ÝÌ, ñòðóêòóðíûìè è êëåòî÷íûìè èññëåäîâàíèÿìè8,14,28-30.



Figure 2. Classical cadherins from adhesive dimers by exchange of the N-terminal ß-strand. (a) A classical cadherin trans dimer is shown as a ribbon diagram in two orthogonal orientations; one protomer is shown in blue, one in orange (from pdb-ID: 3Q2W). Membrane distal EC1 domains overlap and exchange N-terminal ß-strands (expanded view). Note that substantial O- and N-linked glycosylation (magenta and green spheres, respectively) is found on extracellular domains on EC2-4, but not on adhesive EC1 domains. Ca2+ ions are shown as green spheres. (b) The adhesive mechanism of classical cadherins is an example of 3D domain swapping. EC1 domains are shown for the monomer and the dimer (ribbon representation). The swapping element, residue Trp2 (side chain depicted as spheres), has an identical residue environment in the monomer (left panel) and the 'strand-swapped' dimer (right panel). Adapted with permission from [8]. (c) Ribbon presentations of strand-swapped EC1 domains of type I E-cadherin (pdb-ID: 2QVF), type II cadherin-11 (pdb-ID: 2A4E) and VE-cadherin (pdb-ID: 3PPE). Residues characteristic of the adhesive interfaces of each subfamily are depicted as sticks. In type I cadherins, residue Trp2 in domain EC1 is swapped between binding partners. In type II cadherins, two Trp residues, Trp2 and Trp4, are exchanged, and, in addition, hydrophobic interactions occur between conserved residues Phe8, Ile10 and Tyr13 giving rise to an extended interface. VE-cadherin exchanges Trp2 and Trp4 like type II cadherins, but the interface is limited to the apex of the domain, as in type I cadherins.

Îáìåí β-íèòåé íàáëþäàåòñÿ ó êëàññè÷åñêèõ êàäãåðèíîâ íàïð., ïðè ìåõàíèçìå '3D domain swapping' áåëêîâîãî âçàèìîäåéñòâèÿ [31], ïðè êîòîðîì äîìåí îáìåíà (A*-íèòü) ìîæåò ïðèñòûêîâàòüñÿ â ñâîé ñîáñòâåííûé êàðìí, ÷òîáû ñôîðìèðîâàòü 'çàêðûòûé' ìîíîìåð (Figure 2b, left panel) èëè ìîæåò ïðèñòûêîâàòüñÿ â êàðìàí EC1 äîìåíà ïàðòíåðà, ÷òîáû ñôîðìèðîâàòü swapped äèìåð (Figure 2b, right panel). Îáÿçàòåëüíîé ñòóïåíüþ ïðè ïåðåõîäå îò çàêðûòîãî ìîíîìåðà ê îáìåííîìó äèìåðó ÿâëÿåòñÿ îòêðûòîå ñîñòîÿíèå ìîíîìåðà, ïðè êîòîðîì îáìåííûé äîìåí, A* íèòü, îòñòûêîâûâàåòñÿ, ÷òî äåëàåò âîçìîæíûì îáðàçîâàíèå äèìåðà ìåæäó äâóìÿ îòêðûòûìè ìîíîìåðàìè. Âàæíî, ÷òîáû îáìåííûé äîìåí ðàñïîëàãàëñÿ â òåñíîé áëèçè ñî ñõîäíûì îñòàòêîì â 'çàêðûòîì' ìîíîìåðå è îáìåííîì äèìåðíîì ñîñòîÿíèè. Ñëåäîâàòåëüíî, çàêðûòàÿ ìîíîìåðíàÿ ôîðìà ìîæåò áûòü, êàê ïîëàãàþò, êîíêóðåíòíûì èíãèáèòîðîì äëÿ îáìåííîãî (swapped) äèìåðà. Ýòà êîíêóðåíöèÿ îòâåòñòâåííà çà ñëàáîå ñðîäñòâî ñâÿçûâàíèÿ êëàññè÷åñêèõ êàäãåðèíîâ [32], è íåîáõîäèìî, ÷òîáû ñóùåñòâîâàëè ñòðóêòóðíûå ðàçëè÷èÿ, êîòîðûå ñòàáèëèçèðóþò äèìåð è/èëè äåñòàáëèçèðóþò ìîíîìåð, ÷òîáû óïðàâëÿòü äèìåðèçàöèåé.
Ñðàâíåíèå äîìåíîâ êàäãåðèíà, êîòîðûå ó÷àñòâóþò â îáìåíå (swapping) íèòåé (EC1 äîìåíû êëàññè÷åñêèõ êàäãåðèíîâ) ñ non-swapping äîìåíàìè êàäãåðèíîâ (EC2-5) âûÿâèëî ìíîãî÷èñëåííûå ôàêòîðû, êîòîðûå ñïîñîáñòâóþò îáðàçîâàíèþ äèìåðîâ èç îáìåííûõ íèòåé [33]. Swapping äîìåíû êàäãåðèíîâ, êàê áûëî óñòàíîâëåíî, èìåþò óêîðî÷åííóþ β-A íèòü, â äîïîëíåíèå ê çàêîíñåðâèðîâàííîìó òðèïòîôàíó â ïîçèöèè 2, êîòîðûé çàìåùàåòñÿ ôåíèëàëàíèíîì â äð. EC äîìåíàõ. Îñòàòîê ãëþòàìàòîâîé êèñëîòû (Glu11) â îñíîâàíèè A íèòè êîîðäèíèðóåò Ca2+ âî âñåõ êëàññè÷åñêèõ êàäãåðèíàõ è çàêðåïëÿåò A íèòü íà îáîèõ êîíöàõ - â îñíîâàíèè ñ ïîìîùüþ Ca2+ ñâÿçûâàíèÿ ñ Glu11 è íà N êîíöå ïóòåì ñòûêîâêè ñ ïîìîùüþ Trp2 - âûçûâàÿ íàòÿæåíèå óêîðî÷åííîé A íèòè. Ýòî, ñ âîþ î÷åðåäü, äåñòàáèëèçèðóåò çàêðûòûé ìîíîìåð è òåì ñàìûì ñïîñîáñòâóåò îáðàçîâàíèþ swapped äèìåðà, â êîòîðîì ýòî íàòÿæåíèå óñòðàíÿåòñÿ34-36. Ïðèðîäíûé ìîíîìåðíûé non-swapping EC2 äîìåí áûë óñïåøíî 'ïðåâðàùåí' EC1-ïîäîáíûé äîìåí, ñïîñîáíûé ê swap-ñâÿçûâàíèþ ïóòåì âíåñåíèÿ òî÷êîâûõ ìóòàöèé, ïîäòâåðäèâ òåì ñàìûì ýòîò ìåõàíèçì [34].
Èíòåðåñíî, ÷òî õîòÿ íàòÿæåíèå â 'çàêðûòîì' ìîíîìåðå ñïîñîáñòâóåò îáðàçîâàíèþ swapped äèìåðà, èçáèðàòåëüíîå äàâëåíèå òàêæå âîçíèêàåò, ÷òîáû óäåðæàòü ñëàáîå àäãåçèâíîå ñâÿçûâàíèå. Ó òèïà I êëàññè÷åñêîãî êàäãåðèíà EC1 ïîñëåäîâàòåëüíîñòü âêëþ÷àåò çàêîíñåðâèðîâàííûé Pro5-Pro6 ìîòèâ, êîòîðûé ïðåäóïðåæäàåò îáðàçîâàíèå ïîñòîÿííûõ âîäîðîäíûõ ñâÿçåé β-ñëîÿ ìåæäó EC1 äîìåíàìè êàäãåðèíîâ ó àäãåçèâíûõ äèìåðîâ. Åñëè diproline ìîòèâ ìóòàíòåí ïî àëàíèíó â E- è N-êàäãåðèíàõ, òî ñðîäñòâî äèìåðîâ óâåëè÷èâàåòñÿ [34] è â ïðîòèâîïîëîæíîñòü äèêîãî òèïà àíàëîãàì [37] (see below), ñðîäñòâî äèìåðèçàöèè ìóòàíòíîãî N- è E-cadherin îêàçûâàåòñÿ íåîòëè÷èìûì îò íîðìû. Êðèñòàëëè÷åñêèå ñòðóêòóðû ýòèõ ìóòàíòîâ îáíàðóæèâàþò íåïðåðûâíûå â β -íèòè âîäîðîäíûå ìîñòèêè ìåæäó A íèòÿìè EC1 äîìåíîâ ïàðòíåðîâ, ýòî îáúÿñíÿåò ïîòåðþ ñïåöèôè÷íîñòè ñâÿçûâàíèÿ [34]. Ìîòèâ diproline, ïî-âèäèìîìó, íóæäàåòñÿ â ñòðóêòóðíîì ýëåìåíòå, ëåæàùåì â îñíîâå äèôôåðåíöèàëüíîãî ñðîäñòâà ñâÿçûâàíèÿ N- è E-êàäãåðèíà.
Âñå êëàññè÷åñêèå êàäãåðèíû ïîçâîíî÷íûõ èñïîëüçóþò ñõîäíûé ìåõàíèçì îáìåíà íèòåé äëÿ ôîðìèðîâàíèÿ àäãåçèâíûõ äèìåðîâ; îäíàêî, èíòåðôåéñû, îáíàðóæèâàåìûå â êðèñòàëëè÷åñêèõ ñòðóêòóðàõ òèïà I è òèïà II êàäãåðèíîâ îòëè÷àþòñÿ (Figure 2c). Àäãåçèâíûé èíòåðôåéñ òèïà I êàäãåðèíîâ îãðàíè÷åí ðåãèîíîì êàðìàíà âáëèçè âåðõóøêè EC1 (Figure 2c, left panel) è ðåãèîíîì ïàðòíåðñêîé A* íèòè, êîòîðûé âêëþ÷àåò çàêðåïëÿþùèé îñòàòîê Trp2. Íàïðîòèâ, òèïà II êàäãåðèíû, îáíàðóæèâàþò îáìåí äâóìÿ òðèïòîôàíîâûìè îñòàòêàìè, Trp2 è Trp4. Áîëåå òîãî, äèìåðíûé èíòåðôåéñ ó ÷ëåíîâ ñåìåéñòâà òèïà II ðàñïðîñòðàíÿåòñÿ íà âåñü èíòåðôåéñ EC1 äîìåíà, èñïîëüçóÿ êîíñåðâàòèâíûå ãèäðîôîáíûå îñòàòêè â ïîçèöèè 8, 10 è 13 (Figure 2c, middle panel) [26]. Èíòåðåñíî, ÷òî VE-cadherin, îòëè÷àþùèéñÿ îò êëàññè÷åñêîãî êàäãåðèíà è ÿâëÿþùèéñÿ êðèòè÷åñêèì äëÿ àäãåçèè â ñîñóäèñòîì ýíäîòåëèè [38], ñìàçûâàåò îïðåäåëåíèå èíòåðôåéñîâ òèïà I è òèïà II êàäãåðèíîâ. Êàê è â ñëó÷àå òèïà II êàäãåðèíîâ VE-cadherin ñòûêóåò Trp2 è Trp4 ñ ãèäðîôîáíûì êàðìàíîì ñâîåãî ïàðòíåðà, íî ëèøåí ãèäðîôîáíîãî âçàèìîäåéñòâèÿ â îñòàëüíîé ÷àñòè äîìåíà EC1 (Figure 2c, right panel) è ïîýòîìó èìååò â öåëîì ðàñïîëîæåíèå äèìåðà áîëåå ñõîäíûì ñ òàêîâûì ó òèïà I êàäãåðèíîâ [27].
Ñïåöèôè÷íîñòü ãîìîôèëüíîãî ñâÿçûâàíèÿ êëàññè÷åñêèõ êàäãåðèíîâ íà êëåòî÷íîì óðîâíå óïðàâëÿåòñÿ ñ ïîìîùüþ EC1, êàê áûëî óñòàíîâëåíî, â ýêñïåðèìåíòàõ ïî ïåðåòàñêèâàíèþ äîìåíîâ26,39-42, ïîäòâåðæàÿ, ÷òî ðàçëè÷èÿ â èíòåðôåéñå strand-swapping ìîäóëèðóþò ñïåöèôè÷íîñòü. Òèïà I êàäãåðèíû â öåëîì íå ñîåäèíÿþòñÿ ñ ñ òèïîì II êàäãåðèíàìè 8,26,37,43 , ýòî ñâÿçàíî ñ ñóùåñòâåííûìè ðàçëè÷èÿìè â êàíîíè÷åñêîì àäãåçèâíîì èíòåðôåéñå ñòðóêòóðû â êàæäîì ïîäñåìåéñòâå. Èíòåðåñíî, ÷òî êëàññè÷åñêèå êàäãåðèíû âçàèìîäåéñòâóþò áåñïîðÿäî÷íî âíóòðè ïîäñåìåéñòâà, ÷òî ñîãëàñóåòñÿ ñî çíà÷èòåëüíûì ñõîäñòâîì îáëàñòè èíòåðôåéñà ìåæäó èíäèâèäóàëüíûìè ÷ëåíàìè26,37,43,44. Ò.î., âíóòðè ïîäñåìåéñòâ êàäãåðèíû èñïîëüçóþò êàê ãîìîôèëüíûå, òàê è ãåòåðîôèëüíûå ñâîéñòâà ñâÿçûâàíèÿ, êîòîðûå êîìáèíèðóþò, ÷òîáû âûçûâàòü ãîìîôèëüíûå àãðåãàöèè êëåòîê, ýêñïðåññèðóþùèõ êàäãåðèíû [37].

Speed dating: the X-dimer intermediate


Îáðàçîâàíèå strand-swapped äèìåðîâ íóæäàåòñÿ â ïðåîáðàçîâàíèè ñêëàäîê êàæäîãî ïàðòíåðñêîãî ïðîìîòîðà, ÷òîáû ïåðåéòè îò 'çàêðûòîé' ìîíîìåðíîé ôîðìû(Figure 2b, left panel) ê 'îòêðûòîé' äèìåðíîé ôîðìå (Figure 2b, right panel). Ýòî êîíôîðìàöèîííîå èçìåíåíèå ìîæåò äåëàòü äèìåðèçàöèþ êèíåòè÷åñêè íåáëàãîïðèÿòíîé ïîñêîëüêó ýòî âçàèìîïðåâðàùåíèå ìîæåò ïðîèñõîäèòü â òå÷åíèå äëèòåëüíîãî ïåðèîäà âðåìåíè ó äð. áåëêîâ, êîòîðûå ó÷àñòâóþò â 3D äîìåíîâîì swapping [31], íî ýòî ñâÿçûâàíèå ïðîèñõîäèò áûñòðî ó ó êàäãåðèíîâ35,37,45. Áûëè ïðåäëîæåíû äâà àëüòåðíàòèâíûõ ìåõàíèçìà äëÿ îáúÿñíåíèÿ êèíåòèêè âçàèìîäåéñòâèÿ êàäãåðèíîâ: 'selected fit', ïðè êîòîðîì êàäãåðèíîâûå ìîíîìåðû ñóùåñòâóþò â ðàâíîâåñèè ìåæäó îòêðûòîé è çàêðûòîé ôîðìàìè, à äèìåðèçàöèÿ âîçíèêàåò â ðåçóëüòàòå ñòîëêíîâåíèÿ îòêðûòûõ ìîíîìåðîâ; è 'induced fit', ïðè êîòîðîì êàäãåðèíîâûå ìîíîìåðû ñíà÷àëà îáðàçóþò non-swapped ïðîìåæóòî÷íûå îáðàçîâàíèÿ- 'encounter complex' - êîòîðûé ñíèæàåò ýíåðãèþ àêòèâàöèè, íåîáõîäèìóþ äëÿ îáìåíà íèòåé [46]. Íåäàâíî ýêñïåðèìåíòû ïî fluorescence resonance energy transfer (FRET) îäèíî÷íûõ ìîëåêóë ïðåäîñòàâèëè äîêàçàòåëüñòâà ñóùåñòâîâàíèÿ êîìïëåêñà ñîóäàðåíèÿ (encounter complex) â E-cadherin, ñâèäåòåëüñòâóÿ â ïîëüçó èíäóöèðîâàííîãî fit ïóòè äëÿ âçàèìîäåéñòâèÿ, îáåñïå÷èâàåìîãî êëàññè÷åñêèìè êàäãåðèíàìè [47]. Åñëè îáìåí íèòÿìè óñòðàíÿëñÿ ñ ïîìîùüþ ìóòàöèè Trp2 íà Ala, òî äèìåðû âñ¸ åù¸ ôîðìèðîâàëèñü ìåæäó EC1 äîìåíàìè, ïðè ñëåãêà èçìåíåííûõ FRET ðàññòîÿíèÿõ ïî ñðàâíåíèþ ñî swapped äèìåðàìè, óêàçûâàÿ òåì ñàìûì íà ñóùåñòâîâàíèå non-swapped äèìåðíûõ ôîðì. Êðîìå òîãî, ýêñïåðèìåíòû ñ èñïîëüçîâàíèåì atomic force microscope (AFM) ïîêàçàëè, ÷òî non-swapped ìóòàíòíûå äèìåðû ÿâëÿþòñÿ áîëåå ñëàáûìè, ÷åì strand-swapped äèêîãî òèïà äèìåðû, ýòî ýíåðãåòè÷åñêè ñîãëàñóåòñÿ ñ ðîëüþ èõ â êà÷åñòâå ïðîìåæóòî÷íîãî ñâÿçûâàíèÿ [47].
Êðèñòàëëîãðàôè÷åñêèå èññëåäîâàíèÿ strand swap-íàðóøåííûõ êàäãåðèíîâûõ ìóòàíòîâ âûÿâèëè ìîëåêóëÿðíûå äåòàëè ýòîãî encounter êîìïëåêñà [48]. Äëÿ ìíîãî÷èñëåííûõ swapping-íåêîìïåòåíòíûõ ìóòàíòîâ, îáíàðóæèâàëèñü äèìåðû ñ èíòåðôåéñîì, ñîñðåäîòî÷åííûì âîêðóã EC1-EC2 ìåæäîìåíîâîãî ëèíêåðà è âåðõóøêè EC2 (Figure 3, middle panel). Ýòà ñòðóêòóðà òåïåðü îáîçíà÷àåòñÿ êàê 'X-dimer', èç-çà îòíîñèòåëüíîé îðèåíòàöèè âçàèìîäåéñòâóþùèõ ïðîìîòîðîâ, íàïîìèíàþùèõ ïî ôîðìå 'X' (Figure 3). X-äèìåð íå íóæäàåòñÿ â ïåðåñòðîéêå ñêëàäîê (refolding) äëÿ ñâîåãî âçàèìîäåéñòâèÿ, ýòî íàäåëÿåò åãî êèíåòèêîé áûñòðîãî ñâÿçûâàíèÿ. Âàæíî, ÷òî ïîçèöèè X-äèìåðîâ A íèòåé êàæäîãî ïðîìîòîðà ïàðàëëåëüíû äð. äð. â òåñíîé áëèçîñòè, êàê åñëè áû îíè áûëè ñáàëàíñèðîâàíû äëÿ îáìåíà (swap) [48]. Ñõîäíàÿ ñòðóêòóðà íàáëþäàåòñÿ â íèòè ìóòàíòîâ, äåôèöèòíûõ ïî îáìåíó, ó òèïà II cadherin-6 [48].



Figure 3. Strand-swapped adhesive dimers of classical cadherins form through a non-swapped intermediate. E-cadherin monomers (orange and blue ribbon diagrams (left panel); only EC1-2 shown for clarity) associate via an 'X-dimer' interface in which N-terminal strands are not swapped but are closely apposed (middle panel). Swapping of strands leads to formation of mature strand-swapped dimers (right panel). Assembly and disassembly of swapped dimers is likely to proceed via the same pathway. Protomers shown as orange and blue ribbon diagrams with only EC1-2 domains shown for clarity.

Ðîëü X-äèìåðà â êà÷åñòâå encounter êîìïëåêñà ïîäòâåðæäàåòñÿ íàáëþäåíèåì, ÷òî ìóòàöèè, ïðåäíàçíà÷åííûå ïðåäóïðåæäàòü îáðàçîâàíèå X-äèìåðîâ, íî ñîõðàíÿþùèå îáìåí íèòåé èíòàêòíûì, ñóùåñòâåííî çàìåäëÿþò ñêîðîñòü ñâÿçûâàíèÿ E-cadherin è cadherin-6.  ÷àñòíîñòè, íå íàáëþäàåòñÿ äèìåðèçàöèè ïðè short term SPR ìåòîäå, íî îòñóòñòâóåò ïîòåðÿ ñðîäñòâà ïðè äîëãîâðåìåííûõ àíàëèòè÷åñêèõ ýêñïåðèìåíòàõ ñ óëüòðàöåíòðèôóãèðîâàíèåì [48]. Áîëåå òîãî, â îòëè÷èå îò áåëêîâ äèêîãî òèïà, X-äèìåðíûå ìóòàíòíûå ìîíîìåðû è äèìåðû ìîãóò ïåðåéòè â ñòàáèëüíûå ìîíîìåðíûå è äèìåðíûå âèäû, óêàçûâàÿ íà ìåäëåííóþ ñêîðîñòü îáìåíà ìåæäó ýòèìè äâóìÿ ôîðìàìè [48].  íåîïóáëèêîâàííîé ðàáîòå ìû óñòàíîâèëè ñõîäíóþ ñòðóêòóðó è ïîâåäåíèå ñâÿçûâàíèÿ X-äèìåðà äëÿ N-cadherin è êðîìå òîãî, ìóòàöèþ èíòåðôåéñà ïðåäïîëàãàåìîãî X-äèìåðà N-cadherin, äåìîíñòðèðóþùóþ èñ÷åçíîâåíèå àêòèâíîñòè ïî ìåæêëåòî÷íîé àãðåãàöèè [49]. Ïîäîáíî encounter êîìïëåêñó, íàáëþäàåìîìó â FRET ýêñïåðèìåíòàõ, X-äèìåðû áûëè íàéäåíû, êàê îáëàäàþùèå ñëàáûì ñðîäñòâîì ê ñâÿçûâàíèþ ïî ñðàâíåíèþ ñ îáìåííûìè äèìåðàìè äèêîãî òèïà [48]. òðàíñôèöèðîâàííûõ ýïèòåëèàëüíûõ êëåòêàõ, êàäãåðèíîâûå X-äèìåðíûå ìóòàíòû ôîðìèðóþò ÷ðåçâû÷àéíî ñòàáèëüíûå ìåæêëåòî÷íûå ñîåäèíåíèÿ [50], ýòî ñîãëàñóåòñÿ ñ áîëåå ìåäëåííîé ñêîðîñòüþ îáìåíà ìîíîìåð-äèìåð, íàáëþäàåìîé â ýêñïåðèìåíòàõ â îòñóòñòâèè êëåòîê [48], õîòÿ ýôôåêòû íà äèññîöèàöèþ äèìåðîâ áûëè ïîä÷åðêíóòû àâòîðàìè. Èòàê, ñîâðåìåííûå äàííûå ñâèäåòåëüñòâóþò â ïîëüçó ìåõàíèçìà, ñîãëàñíî êîòîðîìó X-äèìåðû ôóíêöèîíèðóþò â êà÷åñòâå ïðîìåæóòî÷íûõ îáðàçîâàíèé ïðè ôîðìèðîâàíèè è ðàçáîðêå 'çðåëûõ' àäãåçèâíûõ äèìåðîâ. Ñòðóêòóðíûå è ôóíêöèîíàëüíûå íàáëþäåíèÿ X-äèìåðîâ ó òèïà I E-cadherin è îòíîñèòåëüíî óäàëåííîãî òèïà II cadherin-6 (34% èäåíòè÷íîñòè â EC1-EC2), âìåñòå ñ ïàòòåðíàìè êîíñåðâàöèè ïîñëåäîâàòåëüíîñòè îñòàòêîâ èíòåðôåéñà [17], ïîäòâåðæäàþò, ÷òî ìåõàíèçì X-äèìåðîâ ìîæåò áûòü îáùèì äëÿ ÷ëåíîâ äâóõ ïîäñåìåéñòâ êëàññè÷åñêèõ êàäãåðèíîâ ó ïîçâîíî÷íûõ .
Èíòåðåñíî, ÷òî T-cadherin, îòëè÷àþùèéñÿ îò êëàññè÷åñêèõ êàäãåðèíîâ, çàêðåïëåí íà ïëàçìàòè÷åñêîé ìåìáðàíå ïîñðåäñòâîì glycosylphosphatidyl inositol (GPI) ÿêîðÿ (Box 1), íå îáíàðóæèâàåò îáìåíà íèòÿìè è àäîïòèðóåò X-äèìåðíóþ êîíôîðìàöèþ äëÿ ñâîåãî çðåëîãî èíòåðôåéñà àäãåçèâíîãî ñâÿçûâàíèÿ [51]. ìóòàöèè, çàòðàãèâàþùèå èíòåðôåéñ X-äèìåðîâ â T-cadherin, áûëè íàéäåíû ïî óñòðàíåíèþ åãî ôóíêöèè â ìîäóëÿöèè ðîñòà íåéðèòîâ, òîãäà êàê öåëåíàïðàâëåííûå ìóòàöèè íèòåé äèìåðà, àíàëîãè÷íî òåì, ÷òî óñòðàíÿëè ñâÿçûâàíèå îáìåííûõ íèòåé ó êëàññè÷åñêèõ êàäãåðèíîâ, íå îêàçûâàëè ýôôåêòà íà ôóíêöèîíèðîâàíèå èëè ãîìîäèìåðèçàöèþ T-cadherin [51]. Ýòè òåñíûå ôèëîãåíåòè÷åñêèå âçàèìîîòíîøåíèÿ ñ òèïîì I êëàññè÷åñêèõ êàäãåðèíîâ óêàçûâàþò íà òî, ÷òî T-cadherin ïðåäñòàâëÿåò ñîáîé êëàññè÷åñêèé êàäãåðèí, êîòîðûé ïîòåðÿë ñâîþ ñïîñîáíîñòü ê îáìåíàì. Äð. ðîëè X-äèìåðîâ ïîìèìî ïîäñåìåéñòâà êëàññè÷åñêèõ êàäãåðèíîâ îñòàþòñÿ íåèçâåñòíûìè.

From bonds to junctions


Ìåæêëåòî÷íàÿ àäãåçèÿ â çðåëûõ òêàíÿõ îáåñïå÷èâàåòñÿ ÷àñòè÷íî ñëèï÷èâûìè ñîåäèíåíèÿìè, ãäå ñîáèðàþòñÿ ìíîãî÷èñëåííûå êàäãåðèíîâûå òðàíñ äèìåðû.  ïðèíöèïå, a ìåõàíèçì îòëàâëèâàíèÿ ïàññèâíîé äèôôóçèè, ïîñðåäñòâîì ÷åãî êàäãåðèíû îêàçûâàþòñÿ ñêîíöåíòðèðîâàííûìè â ìåñòàõ ìåæêëåòî÷íûõ êîíòàêòîâ áëàãîäàðÿ ñâîèì àäãåçèâíûì âçàèìîäåéñòâèÿì, ìîæåò îáúÿñíèòü íàêîïëåíèå êàäãåðèíîâ â ìåñòàõ ìåæêëåòî÷íûõ êîíòàêòîâ [52]. Îäíàêî, ìóòàöèè â êðèòè÷åñêîì öèñ èíòåðôåéñå (described below; Figure 4a) êîòîðûå îñòàâëÿþò àäãåçèâíîå ñâÿçûâàíèå èíòàêòíûì, ïîêàçûâàþò, ÷òî ìåõàíèçì äèôôóçèîííîé ëîâóøêè íåäîñòàòî÷åí äëÿ äîñòèæåíèÿ óðîâíÿ êîíöåíòðàöèè, íàáëþäàåìîãî â ìåæêëåòî÷íûõ êîíòàêòàõ äëÿ êàäãåðèíîâ äèêîãî òèïà [17]. Âîçìîæíî, ÷òî ëàòåðàëüíûå èëè öèñ âçàèìîäåéñòâèÿ ñìîãóò îáúÿñíèòü óñèëåííóþ êîíöåíòðàöèþ êëàññè÷åñêèõ êàäãåðèíîâ â ìåñòàõ êëåòî÷íûõ êîíòàêòîâ.



Figure 4. Extracellular structure of adherens junctions formed through cis and trans ectodomain interactions. (a) Selected region of the N-cadherin EC1-5 crystal lattice (blue ribbon presentation; pdb-ID: 3Q2W) showing an array of N-cadherin molecules oriented as if emanating from the same cell membrane and connected by a cis interface formed between the EC1 and EC2 domains of neighboring molecules. (b) Strand-swapped trans dimers form together with cis interactions in the same crystal lattice. trans interactions orient opposing cis arrays approximately perpendicularly such that each cis array (blue) forms trans interactions with multiple opposing cis arrays (orange). (c) The combination of cis and trans interactions enables cadherin ectodomains to form an ordered network that is thought to be the basis for the extracellular architecture of adherens junction. Adapted from [17].

Ïîòåíöèàëüíîå ìåñòî ëàòåðàëüíîãî âçàèìîäåéñòâèÿ, ïî-âèäèìîìó, çàêîíñåðâèðîâàííîå ñðåäè òèïà I cadherins, íàáëþäàëîñü â êðèñòàëëè÷åñêèõ ñòðóêòóðàõ ýêòîäîìåíîâ ïîëíîé äëèíû ó C- [24], N- è E-cadherins [17]. Íåñìîòðÿ íà îáðàçîâàíèå êðèñòàëëîâ, êîòîðûå íåðîäñòâåííû îäèí äð., ïîìèìî àäãåçèâíîãî strand-swap èíòåðôåéñà, âñå òðè ñòðóêòóðû îáíàðóæèâàþò ëàòåðàëüíûé èíòåðôåéñ, îáðàçóåìûé ìåæäó îñíîâàíèåì äîìåíà EC1 îäíîãî ïðîìîòîðà è îáëàñòüþ â áëèçè âåðõóøêè EC2 èç ïàðàëëåëüíîãî ïàðòíåðà (Figure 4a). Êîìáèíàöèÿ öèñ è òðàíñ âçàèìîäåéñòâèé èñïîëüçóåòñÿ êàæäîé ìîëåêóëîé êàäãåðèíà (Figure 4b) è ñîçäàåò ìîëåêóëÿðíûé ñëîé âíóòðè êàæäîãî êðèñòàëëà, êîòîðûé, ñêîðåå âñåãî, ñîîòâåòñòâóåò âíåêëåòî÷íîé ñòðóêòóðå àäãåçèâíîãî ñîåäèíåíèÿ17,24. Îáëàñòü EC1, âîâëåêàåìàÿ â ýòîò öèñ èíòåðôåéñ, ïðîòèâîïîëîæíà strand-swapping ñàéòó , òàê ÷òî öèñ è òðàíñ âçàèìîäåéñòâèÿ ìîãóò ôîðìèðîâàòüñÿ îäíîâðåìåííî äàâàÿ íåïðåðûâíóþ äâóìåðíóþ ðåøåòêó ñ èçìåðåíèÿìè ïî÷òè òàêèìè, êîòîðûå îæèäàþòñÿ äëÿ ñëèï÷èâûõ ñîåäèíåíèé (Figure 4c).
Êîãäà ýêòîäîìåíû êàäãåðèíîâ ñâÿçàíû ñ ïîâåðõíîñòüþ ëèïîñîì â îòñóòñòâèå äð. áåëêîâ, òî cryo-EM àíàëèç âûÿâëÿåò óïîðÿäî÷åííûå ïîõîæèå íà ñîåäèíåíèÿ ñòðóêòóðû, êîòîðûå íàïîìèíàþò ìîëåêóëÿðíûé ñëîé, íàáëþäàåìûé â C- [24], E- è N-cadherin êðèñòàëëàõ [17]. Ýòà ñèñòåìà, à òàêæå ýêñïåðèìåíòû íà êëåòêàõ, áûëè èñïîëüçîâàíû äëÿ òåñòèðîâàíèÿ èäåè, ÷òî öèñ èíòåðôåéñ ëåæèò â îñíîâå ëàòåðàëüíîé ñáîðêè êàäãåðèíîâ â ñëèï÷èâûõ ñîåäèíåíèÿõ. Ìóòàöèè, êîòîðûå íàöåëåíû íà öèñ èíòåðôåéñ E-cadherin (íå íàðóøàþùèå òðàíñ strand-swapped äèìåðèçàöèþ) âñ¸ åù¸ äåëàþò âîçìîæíûì ðåäóöèðîâàííûé óðîâåíü àäãåçèè ìåæäó ëèïîñîìàìè; îäíàêî, óïîðÿäî÷åííûå ñòðóêòóðû âîññòàíàâëèâàåìûõ ñîåäèíåíèé òåðÿþòñÿ [17]. Ñîîòâ., âêëþ÷åíèå ýòèõ ìóòàöèé â ýíäîãåííûå äèêîãî òèïà êëåòî÷íûå ñîåäèíåíèÿ ïðèâîäÿò ê òîìó, ÷òî ýòè ñîåäèíåíèÿ ñòàíîâÿòñÿ íåñòàáèëüíûìè è âðåìåííûìè [17].  êëåòêàõ, ëèøåííûõ ýíäîãåííîãî êàäãåðèíà, öèñ ìóòàíòíûé áåëîê ëîêàëèçóåòñÿ â ìåñòàõ êëåòî÷íîãî êîíòàêòà, íî íåñïîñîáåí ôîðìèðîâàòü êëàñòåðû â ñòðóêòóðû, ïîäîáíûå ñîåäèíåíèÿì [17]. Èòàê, ýòè äàííûå ïîêàçûâàþò, ÷òî öèñ èíòåðôåéñ, èäåíòèôèöèðîâàííûé â ñòðóêòóðíûõ èññëåäîâàíèÿõ, ó÷àñòâóåò â ëàòåðàëüíîé ñáîðêå êàäãåðèíîâûõ òðàíñ äèìåðîâ â ñëèï÷èâûå ñîåäèíåíèÿ. Öèñ îëèãîìåðèçàöèÿ êàäãåðèíîâ â ñëèï÷èâûõ ñîåäèíåíèÿõ ìîæåò îáúÿñíèòü ðàíåå îáíàðóæåííûå íàáëþäåíèÿ ìíîæåñòâåííûõ àäãåçèâíûõ ñîñòîÿíèé ìåæäó ìîíîñëîÿìè cadherins â ýêñïåðèìåíòàõ ïî ñèëàì ìîëåêóëÿðíûõ âçàèìîäåéñòâèé45,53,54, êîòîðûå ïåðâîíà÷àëüíî èíòåðïðåòèðîâàëè êàê ìíîæåñòâåííûå ñîñòîÿíèÿ òðàíñ äèìåðîâ, íî ìîãóò áûòü îáúÿñíåíû êîìáèíàöèÿìè öèñ è òðàíñ âçàèìîäåéñòâèé.
Èíòåðåñíî, ÷òî öèñ âçàèìîäåéñòâèÿ ñëèøêîì ñëàáûå, ÷òîáû áûòü âûÿâëåíû â ýêñïåðèìåíòàõ ïî ñâÿçûâàíèþ â ðàñòâîðàõ (êîòîðûå îãðàíè÷åíû îáíàðóæåíèåì óðîâíÿ ïðèáëèçèòåëüíî â 1 mM) [17], âñ¸ æå, êàê îáñóæäàëîñü âûøå, ïî-âèäèìîìó, èãðàþò êðèòè÷åñêóþ áèîëîãè÷åñêóþ ðîëü. Ýòî íå óäèâèòåëüíî, ïîñêîëüêó ñèëà âçàèìîäåéñòâèÿ ìåæäó áåëêàìè â ðàñòâîðå ìîæåò îòëè÷àòüñÿ ñóùåñòâåííî îò òàêîãî æå âçàèìîäåéñòâèÿ â êîíòåêñòå îãðàíè÷åííîãî ïåðåäâèæåíèÿ, áóäó÷è ñâÿçàííûì ñ ìåìáðàíîé [55].  ñàìîì äåëå, in silico ýêñïåðèìåíòû ïîäòâåðäèëè, ÷òî êîãäà ôîðìèðóþòñÿ òðàíñ ýêòîäîìåíîâûå äèìåðû, òî ãèáêîñòü äðàìàòè÷åñêè ñíèæàåòñÿ, ïîñêîëüêó äâà âçàèìîäåéñòâóþùèõ ïðîòîìåðà òåïåðü ïðèêðåïëåíû äð. ê äð. ïîñðåäñòâîì àäãåçèâíîãî èíòåðôåéñà è, êðîìå òîãî, êàæäûé çàêðåïëåí íà ìåìáðàíå ïðîòèâîïîëîæíîé êëåòêè [55]. Ò.î., êîãäà ôîðìèðóþòñÿ òðàíñ äèìåðû, òî êîíôîðìàöèîííàÿ ãèáêîñòü ñíèæàåòñÿ, ýòî ïîíèæàåò ýíòðîïèéíûå ïîòåðè, àññîöèèðîâàííûå ñ îáðàçîâàíèåì öèñ äèìåðîâ55,56. Ýòà ìîäåëü, â êîòîðîé öèñ ñáîðêà, íåîáõîäèìàÿ äëÿ òðàíñ äèìåðèçàöèè, ä. îáúÿñíèòü íàáëþäåíèÿ, ÷òî êàäãåðèíû íå îáðàçóþò êëàñòåðîâ â îòñóòñòâèå íåîáõîäèìîé ñëèï÷èâîñòè ñ ïðîòèâîïîëîæíîé êëåòêîé, ýêñïðåññèðóþùåé cadherin12,13.
Êðóïíûå êëåòî÷íûå ñëèï÷èâûå ñîåäèíåíèÿ, òàêèå êàê zonula adherens, êîòîðûå îïîÿñûâàþò ýïèòåëèàëüíûå êëåòêè, âûãëÿäÿò ìåíåå ïëîòíûìè, ÷åì äåñìîñîìû (see below), è âïîëíå âîçìîæíî, ÷òî îíè ñîáèðàþòñÿ èç ìíîãî÷èñëåííûõ ñóáäîìåíîâ, êàæäûé èç êîòîðûõ îïðåäåëÿåò ñòðóêòóðó ñëîÿ, îïèñàííóþ âûøå. Ñòðóêòóðà âíåêëåòî÷íîé ðåøåòêè cadherin íàïðàâëåííîãî äåéñòâèÿ, òàê ÷òî äâà òàêèõ ñóáäîìåíà ä. âñòðå÷àòüñÿ â ïîäõîäÿùåé îðèåíòàöèè, ÷òîáû äîñòè÷ü íåïðåðûâíîãî ñëèÿíèÿ. Ôîðìèðóþòñÿ ëè zonula adherens èç íåïðåðûâíûõ ñòðóêòóð èëè êîëëåêöèè îïðåäåëåííûõ òî÷åê, ïîêà íåÿñíî. Ñîçðåâàíèå ýòèõ ñòðóêòóð íóæäàåòñÿ â àêòèâíîñòè öèòîñêåëåòà, êîòîðûå ìîæåò âûïîëíÿòü ðîëü ïî óïðàâëåíèþ èõ ñáîðêîé èç íåáîëüøèõ òî÷åê [57]. Ñåãîäíÿ ìû ñêëîíÿåìñÿ ê ìíåíèþ, ÷òî êëàñòåðû èç íåáîëüøèõ òî÷åê, ñêîðåå âñåãî, àâòîìàòè÷åñêè ñîáèðàþòñÿ ïîñðåäñòâîì ñâîèõ ýêòîäîìåíîâ, êîãäà äâå ñîîòâ. cadherin-ýêñïðåññèðóþùèå êëåòêè ïðèõîäÿò â êîíòàêò è ïîçäíåå îíè ìîãóò èíêîðïîðèðîâàòüñÿ â çðåëûå êðóïíûå ñëèï÷èâûå ñîåäèíåíèÿ çà ñ÷åò öèòîïëàçìàòè÷åñêèõ îòðîñòêîâ. Íåîáõîäèìû äàëüíåéøèå èññëåäîâàíèÿ äëÿ âûÿñíåíèÿ âçàèìîäåéñòâèÿ ìåæäó âíåêëåòî÷íûì è öèòîïëàçìàòè÷åñêèì ìåõàíèçìàìè â ñáîðêå êàäãåðèíîâ.
Õîòÿ òèïà II êëàññè÷åñêèå êàäãåðèíû èìåþò òîò æå ñàìûé àäãåçèâíûé ìåõàíèçì. ÷òî è òèïà I êàäãåðèíû, öèñ èíòåðôåéñ, îïèñàííûé âûøå, íå îáíàðóæèâàåòñÿ â ëþáîé èç ìóëüòèäîìåíîâûõ êðèñòàëëè÷åñêèõ ñòðóêòóð òèïà II êàäãåðèíîâ26,27,48. Òåì íå ìåíåå èìåþòñÿ äîêàçàòåëüñòâà, ÷òî, ïî êðàéíåé ìåðå, íåêîòîðûå ÷ëåíû ñåìåéñòâà òèïà II êàäãåðèíîâ ìîãóò ôîðìèðîâàòü ñòðóêòóðû, ñõîäíûå ñî ñëèï÷èâûìè ñîåäèíåíèÿìè.  ÷àñòíîñòè, ñîåäèíåíèÿ, îáåñïå÷èâàåìûå äèâåðãåíòíûì VE-cadherin, ïî-âèäèìîìó, ñõîäíû ñ òàêîâûìè äëÿ òèïà I êàäãåðèíîâ, îáíàðóæèâàåìûå ñ ïîìîùüþ ÝÌ [58]. Cadherin-11, òèïà II êàäãåðèí, òàêæå íàáëþäàåòñÿ ñîâìåñòíî ñ ëîêàëèçàöèåé p120-catenin, α-catenin è àêòèíîâûõ ôèëàìåíò â ìåæêëåòî÷íûõ êîíòàêòàõ [59], íî ïðåäñòîèò åù¸ âûÿñíèòü, ôîðìèðóþò ëè cadherin-11 è äð. òèïà II êàäãåðèíû ñîåäèíåíèÿ ñ óëüòðàñòðóêòóðàìè, ñõîäíûìè ñ òåìè, ÷òî íàáëþäàþòñÿ ó òèïà I êàäãåðèíîâ. Ò.î., VE-cadherin è âîçìîæíî äð. òèïà II êàäãåðèíû ìîãóò ôîðìèðîâàòü ìåæêëåòî÷íûå ñîåäèíåíèÿ ïîñðåäñòâîì ðàçíûõ ëàòåðàëüíûõ èíòåðôåéñîâ, óæå óñòàíîâëåííûõ.

Non-classical cadherin subfamilies suggest diversity of adhesive mechanism


Desmosomal cadherins


Àíàëèç êîíñåðâàöèè ïîñëåäîâàòåëüíîñòè óêàçûâàåò íà òî, ÷òî êàäãåðèíû äåñìîñîì (Box 1, reviewed in [60]), îñíîâíîé êîìïîíåíò ñîåäèíåíèé äåñìîñîì, òàêæå ñëèïàþòñÿ ïîñðåäñòâîì ìåõàíèçìà ñâÿçûâàíèÿ îáìåííûìè íèòÿìè (strand-swap). Ýòè êàäãåðèíû èìåþò êëàññè÷åñêèé Trp îñòàòîê, çàêîíñåðâèðîâàííûé â ïîëîæåíèè 2 è ãèäðîôîáíûé îñòàòîê, ñîîòâåòñòâóþùèé êàðìàíó äëÿ ñâÿçûâàíèÿ Trp â êëàññè÷åñêèõ êàäãåðèíàõ33,60. Áîëåå òîãî, Trp2 èëè ãèäðîôîáíûé êàðìàí óñòðàíÿþò òðàíñ ñâÿçûâàíèå desmocollin 2 â ýêñïåðèìåíòàõ ïî ïåðåêðåñòíîìó ñöåïëåíèþ [61]. Ïðè nuclear magnetic resonance (NMR) ñòðóêòóðà ôðàãìåíòà EC1 desmoglein-2 (pdb-ID: 2YQG) ÷åëîâåêà îáíàðóæèâàåò äîìåí ñêëàäêó óäèâèòåëüíî ñõîäíóþ ñ òèïà I êëàññè÷åñêèìè êàäãåðèíàìè. Ýòà ñòðóêòóðà ÿâëÿåòñÿ ìîíîìåðíîé è Trp2 ñòûêóåòñÿ ñàì ñ ñîáîé, âîçìîæíî áëàãîäàðÿ âêëþ÷åíèþ 10 îñòàòêîâ, ïðåäøåñòâóþùèõ íàòèâíîìó N êîíöó áëàãîäàðÿ àðòåôàêòó êëîíèðîâàíèÿ. Ñõîäíûå ðàñøèðåíèÿ, êàê èçâåñòíî, èíãèáèðóþò strand-swap äèìåðèçàöèþ â êëàññè÷åñêèõ êàäãåðèíàõ25,48.
Êðèî-ýëåêòðîííàÿ òîìîãðàôèÿ ïðîçðà÷íûõ ñðåçîâ äåñìîñîì èç êîæè ÷åëîâåêà [62] è ýëåêòðîííàÿ òîìîãðàôèÿ ñðåçîâ êîæè ìûøè, âêëþ÷åííûõ â ïëàñòèê [63] âûÿâèëè âíåêëåòî÷íîå ðàñïîëîæåíèå, ñòàâíèìîå ñ òðàíñ äèìåðèçàöèåé ïîñðåäñòâîì EC1 äîìåíîâ. Äåñìîñîìû â êîæå ÷åëîâåêà îáíàðóæèâàëè âûñîêî óïîðÿäî÷åííîå ðàñïîëîæåíèå âî âíåêëåòî÷íîì ðåãèîíå, òîãäà êàê îíè æå â êîæå ìûøåé áûëè äîâîëüíî íåóïîðÿäî÷åíû âîçìîæíî èç-çà ðàçëè÷èé â îáðàáîòêå ïðåïàðàòîâ. Ñðàâíåíèå ñòðóêòóð ýêòîäîìåíîâ C-cadherin [24] ïðè 34 Å ðàçðåøàþùåì cryo-EM êàðòèðîâàíèè äåñìîñîì â êîæå ÷åëîâåêà ïîäòâåðäèëî ìîëåêóëÿðíóþ óïîðÿäî÷åííîñòü ïî ñðàâíåíèþ ñ ëèíåéíûìè 'çàñòåæêàìè', ñôîðìèðîâàííûìè â ðåçóëüòàòå ÷åðåäóþùèõñÿ EC1-îáóñëîâëåííûõ öèñ- è òðàíñ-âçàèìîäåéñòâèé [62] , îòëè÷íûõ îò äâóìåðíîãî ðàñïîëîæåíèÿ ðÿäîâ, íàáëþäàåìûõ äëÿ òèïà I êëàññè÷åñêèõ êàäãåðèíîâ17,24. Àëüòåðíàòèâíî, âîçìîæíîñòü, ÷òî äåñìîñîìíûå êàäãåðèíû ôîðìèðóþò ñõîäíûå àíñàìáëè êàê è êëàññè÷åñêèå êàäãåðèíû, áûëà ïðåäïîëîæåíà, èñõîäÿ èç EM lanthanide ïðîïèòàííûõ äåñìîñîì èç ñåðäöà ìîðñêèõ ñâèíîê [64]. Ïîñêîëüêó àòîìíîå ðàçðåøåíèå äëÿ ñòðóêòóð öèñ è òðàíñ äèìåðîâ äåñìîñîìíûõ êàäãåðèíîâ ïîêà íåäîñòóïíî, äî äàëüíåéøèå ìóòàöèîííûå è ñòðóêòóðíûå èññëåäîâàíèÿ íåîáõîäèìû äëÿ âûÿâëåíèÿ äåòàëüíîãî ìåõàíèçìà ñâÿçûâàíèÿ. Èíòåðåñíî, ÷òî ñâÿçûâàþùèå âçàèìîäåéñòâèÿ ìåæäó desmogleins è desmocollins òàêæå , êàê áûëî óñòàíîâëåíî, îáëàäàþò âûñîêîé ñòåïåíüþ ñïåöèôè÷íîñòè èçîôîðì [61].

Clustered protocadherins


Ñîáðàííûå â êëàñòåðû protocadherins, íàçâàííûå ïîòîìó, ÷òî îíè êîäèðóþòñÿ â òðåõ íîâûõ êëàñòåðàõ ãåíîâ (α, β è γ) ïðåèìóùåñòâåííî ýêñïðåññèðóþòñÿ â ãîëîâíîì ìîçãå ïîçâîíî÷íûõ (Box 1) è ñîñòàâëÿþò áîëüøîå ïîäñåìåéñòâî cadherin. Îäíàêî èõ àäãåçèâíûå ñâîéñòâà èçó÷åíû ïëîõî. Ìíîãî÷èñëåííûå ñ îäíèì äîìåíîì ñòðóêòóðû âûÿâëåíû äëÿ protocadherins (pdb ID: 2EE0, 2YST, 1WYJ, 1WUZ; [65]), íî íè îäíà, ïî-âèäèìîìó, íå ñîäåðæèò ôóíêöèîíàëüíûé àäãåçèâíûé ñàéò ñâÿçûâàíèÿ, êîòîðûé îñòàåòñÿ íåóëîâèìûì. Èññëåäîâàíèÿ àãðåãàöèè òðàíñôèöèðîâàííûõ êëåòîê ïîêàçàëè ñòðîãóþ ñïåöèôè÷íîñòü ãîìîôèëüíîãî ñâÿçûâàíèÿ äëÿ 7 ÷ëåíîâ protocadherin γ-êëàñòåðà [66].  òîé æå ñàìîé ñèñòåìå ýêñïåðèìåíòû ïî ïåðåòàñêèâàíèþ äîìåíîâ ïîêàçàëè, ÷òî ïîñëåäîâàòåëüíûå äîìåíû ñ EC1 ïî EC3 ÿâëÿþòñÿ êðèòè÷åñêèìè äëÿ òðàíñ àäãåçèè è ÷òî èíòåðåñíî äîìåíû EC2 è EC3, êàê áûëî óñòàíîâëåíî, óïðàâëÿþò ñïåöèôè÷íîñòüþ protocadherin ïðè àãðåãàöèè êëåòîê [66]. Î÷åâèäíî, ýòè äîìåíû îáíàðóæèâàþò íàèâûñøåå ðàçíîîáðàçèå ïîñëåäîâàòåëüíîñòåé ñðåäè èíäèâèäóàëüíûõ èçîôîðì protocadherin [66]. Ò.ê. îäèíî÷íûå íåéðîíû ýêñïðåññèðóþò ìíîãèå èçîôîðìû protocadherin67,68, òî ãîìîôèëüíàÿ ñïåöèôè÷íîñòü ýòîãî òèïà ìîæåò ïðèâîäèòü ê íåíîðìàëüíîìó ñïåêòðó ïîòåíöèàëüíîãî êëåòî÷íîãî ñðîäñòâà. Äàëåå áûëî ïðåäïîëîæåíî, ÷òî ìíîæåñòâåííûå èçîôîðìû ìîãóò àññîöèèðîâàòü â âèäå öèñ òåòðàìåðîâ íà òîé æå ñàìîé êëåòî÷íîé ïîâåðõíîñòè, ÷òîáû îáåñïå÷èòü êîìáèíàòîðíóþ ñïåöèôè÷íîñòü [66], õîòÿ ýòî åù¸ íå ïðîâåðåíî.

Large cadherins with many EC domains


Ìíîãî÷èñëåííûå ÷ëåíû ñâåðõñåìåéñòâà cadherin - ó ïîçâîíî÷íûõ è áåñïîçâîíî÷íûõ - ÿâëÿþòñÿ êðóïíûìè áåëêàìè, ñîäåðæàùèìè ìíîæåñòâî EC äîìåíîâ (Box 1). Õîòÿ îòíîñèòåëüíî ìàëî èçâåñòíî î èõ ñâÿçÿõ ìåæäó ñòðóêòóðîé è ôóíêöèåé, ðàííèå èññëåäîâàíèÿ ïðåäïîëàãàëè, ÷òî íåêîòîðûå èç ýòèõ áåëêîâ ïðèíèìàþò ðàñòÿíóòûå êîíôîðìàöèè, òîãäà êàê äð. ìîãóò ôîðìèðîâàòü ñòðóêòóðû áîëåå ïîõîæèå íà óëîæåííûå ãëîáóëÿðíûå 'ñóïåðäîìåíû'. Äâà àòèïè÷íûõ ÷ëåíà ñâåðõñåìåéñòâà, êîòîðûå, ïî-âèäèìîìó, ïðèíèìàþò ðàñòÿíóòóþ êîíôîðìàöèþ, cadherin-23 (27 EC äîìåíîâ) è protocadherin-15 (11 ECs) (Box 1 and Figure I) [69], ñâÿçûâàþò ñòåðåîöèëèè âîëîñêîâûõ êëåòîê ïóòåì îáðàçîâàíèÿ âíåêëåòî÷íûõ ñòðóêòóð, èçâåñòíûõ êàê ïîí÷èêîâûå ñâÿçêè, ñîáèðàþùèåñÿ ñ ïîìîùüþ òðàíñ ãåòåðîôèëüíîãî âçàèìîäåéñòâèÿ ìåæäó öèñ ãîìîäèìåðàìè [6]. Íåäàâíî àòîìíîå ðàçðåøåíèå ñòðóêòóð N-òåðìèíàëüíîãî ôðàãìåíòà ñ EC1-2 äîìåíàìè cadherin-23 áûëî ïîëó÷åíî70,71, êîòîðîå âûÿâèëî äîìåíîâóþ àðõèòåêòóðó î÷åíü ñõîäíóþ ñ òàêîâîé äð. èçâåñòíûõ êàäãåðèíîâ (Figure 5a), à òàêæå ïðèçíàêè, óíèêàëüíûå äëÿ cadherin-23, âêëþ÷àÿ 310 ñïèðàëü â A íèòè, α-ñïèðàëü ìåæäó β íèòÿìè C è D â EC1 è âîçìîæíî äîïîëíèòåëüíûå ñàéò ñâÿçûâàíèÿ êàëüöèÿ, îáîçíà÷åííûé êàê site 0, íà âåðõóøêå EC170,71. N-òåðìèíàëüíûå ôðàãìåíòû cadherin-23 è protocadherin-15 ïðåäñòàâëåííûå EC1-3 äîñòàòî÷íû äëÿ òðàíñ ãåòåðîôèëüíîãî ñâÿçûâàíèÿ, íî íå äëÿ öèñ ãîìîäèìåðèçàöèè6,70,71. Ñòðóêòóðà ãåòåðîêîìïëåêñà èç cadherin-23 è protocadherin-15 è áîëåå äëèííûå ôðàãìåíòû ýêòîäîìåíà íóæäàþòñÿ â èäåíòèôèêàöèè òðàíñ è öèñ èíòåðôåéñà, êîòîðûå ôîðìèðóþò âåðõóøå÷íóþ ñâÿçêó.



Figure 5. Crystal structures of cadherin-23 and Drosophila N-cadherin reveal unique features of atypical cadherins. (a) Structures of mouse cadherin-23 EC1-2, which are involved in adhesive binding to protocadherin-15 (binding domain indicated by brackets in schematic) reveal successive EC domains (ribbon diagram, pdb-ID: 3MVS, 2WHV) with three Ca2+ ions (green spheres) coordinated in the linker region. Uniquely, a Ca2+ binding site was identified at the apex of EC1 (box), referred to as Ca2+ binding site 0. Structural determination of a complex of cadherin-23 with protocadherin-15 will help to identify the heterophilic binding interface. (b) Structures of DN-cadherin EC1-4, which is part of the adhesive interface for homodimerization (EC1-9, bracket in schematic), reveal four consecutive EC domains (ribbon diagram). Interestingly, Ca2+ coordination was found only between domains EC1-2 and EC3-4 and not between EC2-3 (pink arrow). This Ca2+-free linker introduces a 'kink' in the otherwise linear structure. Sequence analysis suggests a second occurrence of a Ca2+-free linker between EC7 and EC8 in the ectodomain of DN-cadherin; this may contribute to folding of the 16 EC domains into a compact form within the intermembrane space of Drosophila adherens junctions.

DN-cadherin è DE-cadherin ó Drosophila melanogaster ÿâëÿåòñÿ îðòîëîãîì êëàññè÷åñêèõ êàäãåðèíîâ, òåì. ÷òî îíè îáåñïå÷èâàþò Ca2+-çàâèñèìóþ ìåæêëåòî÷íóþ àäãåçèþ, èìåþò çàêîíñåðâèðîâàííûé äîìåí ñâÿçûâàíèÿ armadillo â ñâîåì öèòîïëàçìàòè÷åñêîì ðåãèîíå [72], è ôîðìèðóþò ñëèï÷èâûå ñîåäèíåíèÿ ñ ðàññòîÿíèÿìè ìåæäó ìåìáðàíàìè â 20-30 nm, ñõîäíûìè ñ òàêîâûìè ó ìëåêîïèòàþùèõ14,73,74. Îäíàêî îðãàíèçàöèÿ âíåêëåòî÷íîãî äîìåíà î÷åíü îòëè÷íà îò òàêîâîé ó êëàññè÷åñêèõ êàäãåðèíîâ ïîçâîíî÷íûõ: èìåþòñÿ 8 è 16 EC äîìåíîâ ñ ïðåäñêàçàííûìè ïîñëåäîâàòåëüíîñòÿìè, ðàñïîëîæåííûìè òàíäåìíî â DE- è DN-cadherin [75], ñîîòâ., ñîïðîâîæäàåìûå äîìåíàìè epidermal growth factor (EGF)-like è laminin-G (Box 1, Figure I). Ñòðóêòóðû N-òåðìèíàëüíîé ïîðöèè DN-cadherin íåäàâíî áûëè óñòàíîâëåíû [75] è áûëî âûÿâëåíî, ÷òî ñâîáîäíûé îò Ca2+ ëèíêåðíûé ðåãèîí ìåæäó äîìåíàìè EC2 è EC3 âåäåò ê îáðàçîâàíèþ îñòðîãî óãëà ìåæäó äîìåíàìè âî âñåõ òðåõ êðèñòàëëè÷åñêèõ ôîðìàõ, ýòî çàñòàâëÿåò âî âñåì îñòàëüíîì ëèíåéíóþ ñòðóêòóðó EC1-4 ñêëàäûâàòüñÿ âäâîå ('jackknife') (Figure 5b). Áèîèíôîðìàöèîííûé àíàëèç âûÿâèë, ÷òî äð. äëèííûå êàäãåðèíû, òàêèå êàê FAT, FAT-like, Dachsous è CELSR/Flamingo òàêæå ñîäåðæàò ëèíêåðíûå îáëàñòè ìåæäó äîìåíàìè, êîòîðûå ëèøåíû íåêîòîðûõ èëè âñåõ îñòàòêîâ, íåîáõîäèìûõ äëÿ ñâÿçûâàíèÿ Ca2+ [75]. Ýòè íàõîäêè ïîäòâåðæäàþò, ÷òî äëèííûå êàäãåðèíîâûå ýêòîäîìåíû ìîãóò ñêëàäûâàòüñÿ íà ñàìèõ ñåáÿ, äàâàÿ â ðåçóëüòàòå áîëåå êîìïàêòíîå ðàñïîëîæåíèå, ñîâìåñòèìîå ñ ñ îòíîñèòåëüíî íåáîëüøèì ðàññòîÿíèåì ìåæäó ìåìáðàíàìè ñëèï÷èâûõ ñîåäèíåíèé. Îñòàåòñÿ îïðåäåëèòü, äåëàþò ëè ýòè ëèøåííûå Ca2+ ëèíêåðíûå ðåãèîíû ãèáêèìè èëè îíè ìîãóò âíîñèòü ôèêñèðîâàííûé èçãèá, êàê ýòî íàáëþäàåòñÿ â òðåõ êðèñòàëëè÷åñêèõ ñòðóêòóðàõ. Äåëåöèîííûé ìóòàãåíåç êàðòèðîâàë ìèíèìàëüíûé àäãåçèâíûé ñàéò ñâÿçûâàíèÿ DN-cadherin â îáëàñòè EC1-9 äîìåíà. Áåçóñëîâíî ïîòðåáíîñòü â 9 EC äîìåíàõ óäèâèòåëüíî îòëè÷íà îò êëàññè÷åñêèõ êàäãåðèíîâ ïîçâîíî÷íûõ, äëÿ êîòîðûõ âñå àäãåçèâíûå êîíòàêòû îñóùåñòâëÿþòñÿ ïîñðåäñòâîì EC1-EC1 âçàèìîäåéñòâèé. Ñêëàäûâàþùèé âäâîå èçãèá ìåæäó EC3 è EC4 äîìåíàìè - è äð. ïðåäïîëàãàåìûé ìåæäó EC7 è EC8 (Figure 5b) - íàïîìèíàþò ñâåðõñåìåéñòâî àäãåçèâíûõ áåëêîâ Dscam immunoglobulin, êîòîðîå ñêëàäûâàåòñÿ â ñóïåð-äîìåíîâóþ ïëàòôîðìó, êîòîðàÿ ñîäåðæèò ìíîãèå èììóíîãëîáóëèíîâûå äîìåíû äëÿ îñóùåñòâëåíèÿ àäãåçèâíîãî ñâÿçûâàíèÿ [76].

Concluding remarks


The structural basis of the adhesive function of vertebrate classical cadherins is becoming increasingly clear. Adhesive binding between cells uses a trans strand-swapping mechanism that is enabled by a fast-binding intermediate, the X-dimer. Vertebrate classical cadherins on isolated cells diffuse freely in the plasma membrane, but when they are bound by cognate cadherins from a contacting apposed cell, trans binding lowers the entropic penalty for the formation of cis interactions, initiating lateral oligomerization. These early processes depend only on the properties of cadherin ectodomains, yet subsequent events such as junction strengthening clearly involve interactions of the cadherin cytoplasmic region with regulatory and cytoplasmic elements.
The picture is far less clear for other cadherin subfamilies. Only vertebrate desmosomal cadherins – close relatives of the classical subfamilies – contain sequence elements indicative of strand-swap binding. Other members of the cadherin superfamily, including all invertebrate cadherins, seem likely to engage in adhesive binding by other means, and may adopt diverse binding mechanisms. It is remarkable how many different cadherin–cadherin interfaces have already been discovered, revealing a surprising complexity in the interactions of classical cadherins. However, longer cadherins appear to form interfaces through surfaces not yet defined. Moreover, some cadherins are known to bind to other proteins and, although for some of these the structural basis is known (for example E-cadherin binding to NKLRG1 [77] and to internalin [78]), for others, such as integrins [79], we have little structural insight into how such binding occurs. It is clear that, at this stage, our structural understanding is limited to only a small portion of the wider cadherin universe, which appears to exploit the remarkable versatility of the cadherin fold in forming diverse sets of protein–protein interfaces. Progress in our understanding of classical cadherins emphasizes the utility of combining insights from structural, cell biological, biophysical and computational studies. The new mechanistic insights that have been inferred may be applicable to many other classes of adhesion receptors.