Domain organization of ZO proteins and the structure of the SH3-GUK domains of ZO-1. A, domain organization of the ZO MAGUKs. The canonical MAGUK protein binding motifs (PDZ, SH3, and GUK) are separated by “unique” (U) regions of high sequence diversity. TJ binding partners of ZO-1 are mapped above their known interaction domain with the transmembrane strand components labeled in red (reviewed in Ref. 14). Those interactions that are also conserved in ZO-2 and ZO-3 are indicated, and the section of ZO-1 whose structure is presented here is indicated by dashed square brackets. B, ribbon diagram of the ZO-1 SH3 (cyan) and GUK (green) domains. The fifth strand of the SH3 domain and the strand following the GUK domain form the interdomain linkage (pink). The ZO-1 construct used for the structure determination lacked the U5 region; arrow 1 points to the truncation point (orange highlight). The entire structure could be traced with confidence except for 3 residues located at the NMP-binding region at the GUK domain (arrow 2, orange highlight).

Sequence alignment of SH3-GUK modules from human ZO proteins and rat PSD-95. The initial alignment was accomplished using ClustalW and later improved manually. Residues conserved in all four sequences are highlighted in black, and those present in three sequences are highlighted in gray. The secondary structure elements shown above the sequences are based on ZO-1 and maintain the color-coding of Fig. 1B. The boundaries for the residues deleted from the SGΔU5 construct used for the structure determination are indicated. *, the cis-proline residue in PSD-95 (Pro⁷¹⁴) that is responsible for the acute angle between its SH3 and GUK domains. Residues in ZO-1 helix V that were mutated are indicated in orange letters for the triple mutant, with the two additional residues for the pentamutant shown in red.

ZO-1 adopts a more open interdomain conformation relative to PSD-95. A, overlay of the SH3-GUK modules of ZO-1 (cyan and green) and PSD-95 (purple and magenta). The superposition was generated by aligning residues of the SH3 domains. The two strands forming the interdomain linkage are colored pink for ZO-1 and red for PSD-95. When the SH3 domains are aligned, the GUK domains occupy significantly different positions, with the PSD-95 adopting a more closed state. This difference in interdomain angle is attributed to the nature of the residue located between the terminal GUK helix VI (labeled with an asterisk) and β-strand 6. In PSD-95, a cis-proline (Pro⁷¹⁴) forces an acute angle between these secondary structure elements (see inset). ZO-1 has a glutamine residue at this position, allowing for a more open conformation. B, superposition of the SH3 domains. The deleted U5 region (orange trace) in ZO-1 is indicated by an arrow. In PSD-95, despite the presence of the U5 residues, only a section could be traced, with two arrows indicating the break. Note that the sole helix located within the SH3 domain adopts a different angle in ZO-1 and PSD-95 (double-headed gray arrow) relative to the core of the SH3 domain. C, superposition of the GUK domains. PSD-95 has a significantly longer insert at a region called the NMP-binding domain (see Fig. 2), with ZO-1 having only a short loop (the three residues that could not be modeled in this loop are shown in orange). When using the GUK residues for calculating the superposition, the interdomain strands (pink and red for ZO-1 and PSD-95, respectively) do not overlay well (indicated by two arrows). The converse is true when calculating the overlay with SH3 residues (B). Thus, the interdomain strands are an integral part of the SH3 domain.

The GUK domain of ZO-1 is characterized by a positively charged surface. A, a ribbon diagram of ZO-1 with all GUK domain lysine and arginine residues shown. B, electrostatic surface potential at the same orientation as in A. C, view rotated 90° relative to A. D, electrostatic surface potential at the same orientation as in C. A congregation of basic residues occurs at and near helix V of the GUK domain. In C, the basic residues probed by mutagenesis are colored dark blue, and all others are shown in light blue.

Helix V of the GUK domain is important for binding to calmodulin and the U6 motif. A, GST-CaM was immobilized on glutathione beads and incubated with various SG constructs in the presence of 1 mm CaCl₂ and then washed to remove unbound protein. GST-CaM and bound proteins were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. Lane 1, molecular weight markers; lanes 2, 5, 9, and 13, the fraction “bound” to GST-CaM after incubation with SG, the triple charge elimination mutant (TM(A)), the triple charge reversal mutant (TM(D/E)), or the penta-charge reversal mutant (PM(D/E)), respectively. Calmodulin binding to PM(D/E) and, to a lesser extent TM(D/E), is attenuated relative to SG. Lanes 3, 6, 10, and 14, supernatant (unbound fraction) after spinning down the beads. Lanes 4, 8, and 12, controls, in which the proteins were simply incubated with the glutathione beads. The lack of a band shows that there is no interaction between the SG constructs and the beads. Lanes 7, 11, and 15, controls in which GST alone on beads (instead of GST-CaM) was incubated with the SG constructs. This shows that the SG constructs do not interact with GST. The ratio of SG or SG mutants relative to GST-CaM, calculated from the intensity of the bands within the red box, is presented for each condition above the box. B, the U6 region binds SG in cis. Lanes 4, 8, and 12, proteins bound to GST-U6 after incubation with SG in the presence of 1 mm MgCl₂, 1 mm CaCl₂, or EDTA, respectively. The binding of U6 to SG is dependent on divalent metal. Lane 1, molecular weight markers. Lane 2, GST-U6 by itself on the beads. Lanes 3, 7, and 11, controls where the SG was incubated with glutathione beads, showing no nonspecific interactions. Lanes 5, 9, and 13, supernatant (unbound) fraction after the pull-down. About 50% of GST-U6 binds SG in the presence of magnesium and 100% in calcium, but little or none binds in the EDTA-containing condition. Lanes 6, 10, and 14, controls with GST instead of GST-U6. Quantified band ratios are shown above the red box. C, experiment analogous to that in B but with SGΔU5 instead of SG. This demonstrates that the divalent cation-dependent binding of U6 to SG is independent of the U5 region. D, experiment analogous to that in A but with GST-U6 instead of GST-CaM. We see that the charge reversal mutations (lanes 9 and 13) largely abolish the interaction between U6 and SG. The charge elimination mutations (lane 5) only moderately decrease the interaction. E, GST-CaM was immobilized on glutathione beads and incubated with the ZO-1 construct SH3-GUK-U6 (SGU6), SH3-GUK (SG), or SH3-GUK that lacks U5 (SGΔU5). Lanes 1 and 15, marker lanes. Lane 2, GST-CaM bound to the beads. Lanes 3, 7, and 11, control conditions in which the protein, SGU6, SG, or SGΔU5, respectively, was incubated with the glutathione beads; no nonspecific binding was observed. Lanes 4, 8, and 12, the fraction “bound” to GST-CaM after incubation with SGU6, SG, or SGΔU5, respectively. GST-CaM does not pull down SGU6, but it does pull down SG and SGΔU5. Quantified band ratios are shown above the red box. This demonstrates that the binding of calmodulin or the U6 region to the GUK domain of ZO-1 is mutually exclusive. Lanes 5, 9, and 13, supernatant (unbound fraction) after spinning down the beads. Lanes 6, 10, and 14, controls in which GST alone on beads (instead of GST-CaM) was incubated with the SG constructs. This shows that the SG constructs do not interact with GST.

Human ZO-1 adopts a different SH3-GUK interdomain conformation compared with ZO-3. A, surface representation of ZO-1 in which the sequence conservation to ZO-3 is indicated by different colors: green for identical residues, lime for similar residues, and orange for non-conserved residues. An arrow indicates the location of the U5 region. B, an analogous representation rotated 180 degrees. C, superposition of ZO-1 (same color scheme as before) and ZO-3 (gold) based on aligning the SH3 domains. The SH3-GUK interdomain angle is different, with ZO-3 adopting a more open conformation. The U5 region of ZO-3 could not be traced, with the break indicated by two gold arrows. D, superposition of the GUK domains reveals a very similar fold. The NMP-binding loop in ZO-3 could not be traced (arrow 1 points at the break), whereas we could model most of this region in ZO-1 except the orange highlighted stretch. Because the proteins adopt a different interdomain angle, the strands linking the domains do not align well (arrow 2). E, superposition of the SH3 domains shows a nearly perfect overlay. This time the interdomain strands overlay well, indicating that they move as a rigid body with the SH3 domain. Two arrows indicate the break in the U5 region of ZO-3 relative to the break in ZO-1 (orange trace). Note the extra helix in ZO-3 (arrow 2) relative to that of ZO-1.

Presence of the U5 region in ZO-1 does not change the interdomain conformation. A 3.7 Å data set was collected on an SG construct containing the U5 region (SG), and the structure was solved by molecular replacement using the SH3 and GUK as two independent domains. This structure maintains the interdomain positioning as observed in SGΔU5. The color scheme of SG follows that of SGΔU5 but with a lighter shade.