NMR Characterization of Structural and Dynamics Perturbations Due to a Single Point Mutation in Drosophila DLC8 Dimer: Functional Implications † P. M. Krishna Mohan and Ramakrishna V. Hosur* Department of Chemical Sciences, Tata Institute of Fundamental Research Homi Bhabha Road, Mumbai 400 005, India ReceiVed March 27, 2008; ReVised Manuscript ReceiVed April 18, 2008 ABSTRACT: Dynein light chain protein (DLC8), the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions, and cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface can thus be anticipated to play crucial roles in the variety of functions the protein performs. Recent investigations point out that DLC8 also gets phosphorylated at Ser 88, which is located at the extreme C-terminal end. In this background, we investigate here by NMR the effects of a small perturbation by way of a single point mutation, S88A, on the structure, dynamics, and cargo binding efficacy of the DLC8 dimer. We observe that the perturbation travels far away along the sequence from the site of the mutation. This relay has been explained at the atomic level by looking into the packing of the side chains in the crystal structure of the protein. It follows that the interface is highly adaptable, which may account for the versatility of the dimer’s cargo binding ability. Binding studies with a peptide indicate that the mutation compromises binding efficacy. These observations show how remote residues that may not be directly bound to a target can still affect the affinity of the protein to the target. Furthermore, the S88A mutational perturbations seen here in Drosophila DLC8 are dramatically different from those of the same mutation in human DLC8 (also known as DLC1) (Song, C., et al., (2008) J. Biol. Chem, 283, 4004-4013.) which differs from Drosophila DLC8 at only five locations. All of these observations put together highlight the sensitivity of dynein light chain protein to small perturbations, and this would have great functional implications. Cytoplasmic dynein is a microtubule-based multisubunit molecular motor that translocates cargos toward the minus ends of microtubules. Dynein is involved in a wide array of functions including retrograde organelle movement, nuclear migration, miotic spindle alignment and axonal transport (1–5). The heavy chain subunits provide ATPase activity essential for force generation and are also responsible for attaching the motor complex to microtubules. The cargo binding domain comprising intermediate and light chain subunits regulate the binding and transport of a wide variety of cargo molecules. Dynein light chain protein (DLC8 1 ) is the smallest (89 amino acids, 10.3 kDa) among the light chains of the dynein motor complex. At physiological pH, the protein exists as a homodimer but dissociates into monomers below pH 4 (6, 7). The DLC8 dimer consists of two R-helices (R1, residues 15-31; R2, residues 35-50) and 5 -strands (1, residues 6-11; 2, residues 54-59; 3, residues 62-67; 4, residues 72-78; and 5, residues 81-87) (8). The dimer binds the target molecules in an antiparallel -strand fashion through its 3-strand and also forms several contacts with the residues that are present at the dimer interface (8). Furthermore, it has been reported that the monomeric DLC8 protein is not capable of binding any of the target molecules (9). These facts suggest that the topology of the dimer facilitates cargo loading in the dimer. Other than binding to the intermediate chain (IC74) within the dynein complex, DLC8 interacts also with a large number of cellular targets with diverse biological functions. It interacts with neuronal nitric oxide synthase (nNos) (10) proapoptotic BCl-2 family proteins Bim and Bmf (11, 12), postsynaptic scaffold proteins GKAP and gephyrin (13, 14), nuclear protein p53-binding protein 1 (53BP1) (15), and mRNA localization protein Swallow (16). The five-residue K/RXTQT sequence has been recognized as one of the common DLC8 recognition motifs in target peptides (17). Since DLC8 interacts with various proteins, it was presumed that DLC8 acts as a cargo adaptor in the dynein complex to transport various organelles. Recent studies on p21-activated kinase 1 (Pak1), a member of the evolutionarily conserved family of serine/threonine kinases, revealed DLC8 as its physiological interacting substrate with binding sites mapped to amino acids 61-89 and the phosphorylation site at Ser 88 (18). The phospho- rylation of DLC8 by Pak1 plays a regulatory role in tumorigenesis and in macropinocytosis (18–20). Pak1 phos- † We thank the Government of India for providing financial support to the National Facility for High Field NMR at the Tata Institute of Fundamental Research. P.M.K.M. is a recipient of TIFR alumni Association Scholarship (2003-2005) and Sarojini Damodaran inter- national fellowship for career development, supported by the TIFR endowment fund. * To whom correspondence should be addressed. Professor Ra- makrishna V. Hosur, Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India. Tel: 91 22 2278 2488. Fax: 91 22 2280 4610. E-mail: hosur@tifr.res.in. 1 Abbreviations: DLC8, dynein light chain protein; NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum coherence; DTT, dithiothreitol; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-poly acrylamide gel electrophoresis; aa, amino acids; WT, wild type. Biochemistry 2008, 47, 6251–6259 6251 10.1021/bi800531g CCC: $40.75  2008 American Chemical Society Published on Web 05/09/2008