Structural basis of diverse sequence-dependent target recognition by the 8 kDa dynein light chain¹

Abstract

Dyneins are multi-subunit molecular motors that translocate molecular cargoes along microtubules. Otherthan acting as an essential component of the dynein motor complex, the 89-residue subunit of dynein light chain (DLC8) alsoregulates a number of other biological events by binding to various proteins and enzymes. Currently known DLC8 targets includeneuronal nitric oxide synthase; the proapoptotic Bcl-2 family member protein designated Bim; a Drosophila RNA localizationprotein Swallow, myosin V, neuronal scaffolding protein GKAP, and IκBα, an inhibitor of the NFκB transcription factor.The DLC8-binding domains of the various targets are confined within a short, continuous stretch of amino acid residues. However,these domains do not share any obvious sequence homology with each other. Here, the three-dimensional structures of DLC8 complexedwith two peptides corresponding to the DLC8-binding domains of neuronal nitric oxide synthase and Bim, respectively, weredetermined by NMR spectroscopy. Although the two DLC8-binding peptides have entirely different amino acid sequences, both peptidesbind to the protein with a remarkable similar conformation by engaging the symmetric DLC8 dimer through antiparallel β-sheetaugmentation via the β2 strand of the protein. Structural comparison indicates that the two target peptides usedifferent regions within the conformational flexible peptide-binding channels to achieve binding specificity. We have alsore-determined the apo-form solution structure of DLC8 in this work. The structures of the DLC8/target peptide complexes,together with the dynamic properties of the protein, provide a molecular basis of DLC8’s diverse amino acid sequence-dependenttarget recognition.

Introduction

Cytoplasmic dynein is a multi-subunit, minus-end-directed molecular motor. It is involved in a wide range of motile eventsincluding retrograde axonal vesicle transport, membrane trafficking, spindle assembly and orientation, and nuclear migrationHirokawa 1998, Karki and Holzbaur 1999, King 2000, Vallee and Sheetz 1996. The complex consists of two microtubule-binding heavychains containing ATPase and motor activities (dynein heavy chain (DHC), ∼500 kDa), several dynein intermediate chains(DIC, ∼74 kDa) that are involved in cellular targeting, and a group of light intermediate chains (∼52–61 kDa)that are thought to regulate dynein motor activity Hughes et al 1995, Paschal et al 1992. Cytoplasmic dynein also contains several light chains (DLCs,∼8–22 kDa), whose biological functions are just beginning to be uncovered King 2000, King et al 1996a, King et al 1996b.

Unlike all other subunits that are stoichiometrically associated with the dynein complex, only a minor portion of the 8-kDasubunit of dynein light chain (DLC) (DLC8, comprising 89 amino acid residues with an actual mass of 10.3 kDa) is directlyassociated with dynein (King et al., 1996a). The majority of DLC8 is present in thecytoplasm in a non-microtubule-associated form. DLC8 is ubiquitously expressed in different cell types and is highly conservedthroughout evolution, implying that the protein is likely to be a multifunctional regulatory protein Jaffrey and Snyder 1996, Tochio et al 1998. Genetic studies showed that DLC8 isessential for the motor activity of the dynein complex since a DLC8 null mutation inhibits nuclear migration in Aspergillusnidulans (Beckwith et al., 1998). In Drosophila, partial loss-of-functionmutations of DLC8 cause morphogenetic defects in bristle and wing development, female sterility, and disruption of sensory axonprojections Dick et al 1996, Phillis et al 1996,and complete loss-of-function of DLC8 is embryonic lethal via apoptotic events (Dick et al., 1996). Other than binding to molecular motors such as dynein and myosin V (Benashski et al., 1997), DLC8 was also found to bind to a number of other proteins and enzymes whichpossess diverse biological functions. For example, DLC8 binds to a 17-residue peptide fragment immediately preceding the oxygenasedomain of neuronal nitric oxide synthase Fan et al 1998, Jaffrey and Snyder 1996, although the biological significance of this interaction is not entirely clear. The protein wassubsequently named PIN for the protein inhibitor of neuronal nitric oxide synthase (nNOS) (to avoid nomenclature confusion, we useDLC8 in this work in order to be consistant with the more frequently used name in the literature). Yeast two-hybrid screeningidentified that DLC8 can interact with the N-terminal regulatory domain of IκBα, an inhibitor of the NFκB transcriptionfactor (Crepieux et al., 1997). Recently, DLC8 was found to specifically interact with Bim, anewly discovered proapoptotic Bcl-2 family protein Puthalakath et al 1999, O’Connor et al 1998. In healthy cells, formation of the DLC8/Bim complex sequesters themajority of Bim to the microtubule-associated dynein complex. Upon apoptotic stimulation, the DLC8/Bim complex is released fromthe microtubule, and Bim is therefore freed to neutralize the anti-apoptotic activity of Bcl-2 by forming a Bim/Bcl-2heterodimer (Puthalakath et al., 1999). In Drosophila, interaction between DLC8 andSwallow was shown to be critical for asymmetric RNA localization (Schnorrer et al., 2000).DLC8 was also shown to interact with the neuronal scaffold protein, GKAP. Formation of the myosin V/DLC8/GKAP ternarycomplex may play important roles in the trafficking of neuronal signalling complexes (Naisbitt et al., 2000). Inspection of the amino acid sequences of several DLC8 target proteins (e.g. nNOS, Bim, GKAP, and IκBα)reveals that the DLC8-binding regions do not share obvious amino acid sequence homology, which suggests that DLC8 is capable ofbinding to different targets with diverse amino acid sequences. However, the molecular mechanism of DLC8’s divergent sequencerecognition is essentially unknown partially due to the lack of detailed biochemical and structural studies of suchinteractions.

Here, we report the high-resolution solution structures of DLC8 complexed with its binding domains derived from nNOS (thenNOS peptide, comprising residues Met228 to Lys245 of nNOS) and Bim_L(48 to 56), respectively Fan et al 1998, Puthalakath et al 1999. The solution structure of DLC8complexed with the nNOS peptide is essentially identical to the recently solved X-ray structure of the protein complexed with ashorter peptide derived from nNOS (Liang et al., 1999). Detailed comparison of the three-dimensional structures of thecomplexes provides insights into the molecular basis of DLC8’s diverse sequence-dependent target recognition.