Stability and specificity of heterodimer formation for the coiled-coil neck regions of the motor proteins Kif3A and Kif3B: the role of unstructured oppositely charged regions

Abstract

We investigated the folding, stability, and specificity of dimerization of the neck regions of the kinesin-like proteins Kif3A (residues 356-416) and Kif3B (residues 351-411). We showed that the complementary charged regions found in the hinge regions (which directly follow the neck regions) of these proteins do not adopt any secondary structure in solution. We then explored the ability of the complementary charged regions to specify heterodimer formation for the neck region coiled-coils found in Kif3A and Kif3B. Redox experiments demonstrated that oppositely charged regions specified the formation of a heterodimeric coiled-coil. Denaturation studies with urea demonstrated that the negatively charged region of Kif3A dramatically destabilized its neck coiled-coil (urea1/2 value of 3.9 m compared with 6.7 m for the coiled-coil alone). By comparison, the placement of a positively charged region C-terminal to the neck coiled-coil of Kif3B had little effect on stability (urea1/2 value of 8.2 m compared with 8.8 m for the coiled-coil alone). The pairing of complementary charged regions leads to specific heterodimer formation where the stability of the heterodimeric neck coiled-coil with charged regions had similar stability (urea1/2 value of 7.8 m) to the most stable homodimer (Kif3B) with charged regions (urea1/2 value of 8.0 m) and dramatically more stable than the Kif3A homodimer with charged regions (urea1/2, value of 3.9 m). The heterodimeric coiled-coil with charged extensions has essentially the same stability as the heterodimeric coiled-coil on its own (urea1/2 values of 7.8 and 8.1 m, respectively) suggesting that specificity of heterodimerization is driven by non-specific attraction of the oppositely unstructured charged regions without affecting stability of the heterodimeric coiled-coil.

Figure 1

Top panel shows a schematic of the kinesin-like heterotrimeric motor protein Kif3A/Kif3B/KAP3. This protein consists of two different polypeptide chains, Kif3A and Kif3B, which resemble conventional kinesin heavy chains. Each polypeptide chain contains a N-terminal globular motor domain, followed by a potential neck coiled-coil region, a highly charged hinge region, a coiled-coil stalk region and the C-terminal tail region (which binds to an accessory unit, the kinesin-like associated protein KAP3). Bottom panel shows the amino acid sequences of the complementary charged regions in Kif3A and Kif3B. Shown are the charged regions, residues 378–416 and 373–411, which comprise the non-homologous regions of Kif3A and Kif3B, respectively. The charged region of Kif3A begins with negatively charged amino acids followed by positively charged amino acids; the analogous region in Kif3B begins with a positively charged region followed by a glycine spacer and then a negatively charged region. CGG represents a flexible linker added N-terminally to each peptide. Ac-denotes N^α-acetyl and -amide denotes C^α-amide.

Figure 2

Circular dichroism (CD) spectra of the complementary charged regions. Panel A depicts the random coil spectra of the non-linked (reduced) peptides denoted P5r, P10r and P5r mixed P10r (1 : 1) denoted P5r/P10r. Panel B shows the random coil spectra of the disulfide-bridged (oxidized) homostranded peptides denoted P5x, P10x and the heterostranded peptide P5/P10x. P5, open squares; P10, open triangles; and P5/P10, closed circles. The peptide sequences are shown in Fig. 3.

Figure 3

Amino acid sequences of the Kif3A and Kif3B peptides used in this study. The native mouse Kif3A and Kif3B sequences, residues 356–416 and 351–411, are shown above the synthetic peptides prepared for this study. The open box around the native sequence indicates the predicted neck α-helical coiled-coil region. The heptad repeat of the coiled-coil is denoted by abcdefg, where positions a and d are typically the non-polar residues (shown in bold) involved in the 3–4 hydrophobic repeat. The boxed regions below the peptide sequences denote the predicted coiled-coil region and the highly negatively charged and positively charged regions. The negatively charged residues and positively charged residues are indicated by a (−) or (+) sign, respectively. CGG represents a flexible linker added N-terminally to each peptide. Ac-denotes N^α-acetyl and -amide denotes C^α-amide. All of the synthetic peptides are aligned to the native sequence except peptides P4 and P9, which consist of coiled-coil regions, residues 356–377 and 351–372, respectively, combined with the positively charged region, residues 403–416, and the negatively charged region, residues 394–411.

Figure 4

Comparison of hetero-two-stranded coiled-coil with homo-two-stranded coiled-coil neck regions. Panel A depicts the CD spectra of the three heptad coiled-coils for the neck regions of Kif3A/Kif3B, Kif3A, and Kif3B (peptides P2/P7, P2 and P7, respectively). Panel B depicts the relative stabilities of the neck region coiled-coils in guanidine hydrochloride. Panel C depicts the relative stabilities of the neck region coiled-coils for the homo-and hetero-two-stranded peptides in urea. Homostranded Kif3A neck region, P2, (open circles); homostranded Kif3B neck region, P7, (open squares); heterostranded Kif3A/ Kif3B neck region, P2/P7, (closed triangles). Peptide sequences are shown in Fig. 3.

Figure 5

The formation of oxidized, heterostranded P1/P6, P3/P8, P4/ P9, and P2/P7 from redox experiments as a function of time. Reaction A represents the redox results of oxidized peptides P1 and P6 (closed triangles in plot). Reactions B and C represent the redox reactions of oxidized P3 with P8 and P4 with P9, respectively (open diamonds and open squares). Reaction D represents the redox reaction of peptide P2 with P7 (open circles). The percent heterostranded peptide was calculated by dividing the integrated area of the hetero-two-stranded HPLC peak by the total integrated area of both the homo- and heterostranded peptides for each time point. Peptide sequences are shown in Fig. 3.

Figure 6

Formation of heterostranded peptides in the presence of NaCl. The dashed line is the theoretical maximum in benign medium. Reaction A represents the redox results of oxidized peptides P1 and P6 (closed triangles). Reaction B represents the result of mixing oxidized peptides P3 and P8 (open diamonds). Reaction C is the result of mixing oxidized peptides P4 and P9 (open squares). Reaction D is the result of mixing oxidized peptides P2 and P7 (open circles). Percent heterostranded peptide was calculated as described for Fig. 5.

Figure 7

Comparison of stabilities of oxidized peptides P3, P8, and P3/ P8: coiled-coils with the first segment of the complementary charged region. Panel A depicts the relative stabilities, in guanidine hydrochloride, for homostranded peptide P3, heterostranded peptide P3/P8, and homostranded peptide P8. The peptides are comprised of the neckregion coiled-coils with the first segment from the complementary charged regions found in Kif3A and Kif3B (Fig. 3) added C-terminally. Panel B depicts the relative stabilities of peptides P3, P3/P8, and P8 in urea. Homostranded peptides P3 and P8 (open circles and open squares, respectively); heterostranded peptide P3/P8 (closed triangles). Peptide sequences are shown in Fig. 3.

Figure 8

Comparison of stabilities of oxidized peptides P1, P6, and P1/P6: coiled-coils with both segments of complementary charged residues. Panel A depicts the relative stabilities of the coiled-coil neck regions for homostranded P1, heterostranded P1/P6, and homostranded P6 in guanidine hydrochloride. The peptides are comprised of the coiled-coils with the full complementary charged regions (Fig. 3) added C-terminally. Panel B depicts the stabilities of the peptides in urea. Homostranded peptides P1 and P6 (open circles and open squares, respectively); heterostranded peptide P1/P6 (closed triangles). Peptide sequences are shown in Fig. 3.