Michael David Clark

All Publications

• The conformational cycle of prestin underlies outer-hair cell electromotility NATURE Bavi, N., Clark, M., Contreras, G. F., Shen, R., Reddy, B. G., Milewski, W., Perozo, E. 2021; 600 (7889): 553-+

  Abstract

  The voltage-dependent motor protein prestin (also known as SLC26A5) is responsible for the electromotive behaviour of outer-hair cells and underlies the cochlear amplifier1. Knockout or impairment of prestin causes severe hearing loss2-5. Despite the key role of prestin in hearing, the mechanism by which mammalian prestin senses voltage and transduces it into cellular-scale movements (electromotility) is poorly understood. Here we determined the structure of dolphin prestin in six distinct states using single-particle cryo-electron microscopy. Our structural and functional data suggest that prestin adopts a unique and complex set of states, tunable by the identity of bound anions (Cl- or SO42-). Salicylate, a drug that can cause reversible hearing loss, competes for the anion-binding site of prestin, and inhibits its function by immobilizing prestin in a new conformation. Our data suggest that the bound anion together with its coordinating charged residues and helical dipole act as a dynamic voltage sensor. An analysis of all of the anion-dependent conformations reveals how structural rearrangements in the voltage sensor are coupled to conformational transitions at the protein-membrane interface, suggesting a previously undescribed mechanism of area expansion. Visualization of the electromotility cycle of prestin distinguishes the protein from the closely related SLC26 anion transporters, highlighting the basis for evolutionary specialization of the mammalian cochlear amplifier at a high resolution.

  View details for DOI 10.1038/s41586-021-04152-4

  View details for Web of Science ID 000724704200001

  View details for PubMedID 34695838

• Electromechanical coupling in the hyperpolarization-activated K+ channel KAT1. Nature Clark, M. D., Contreras, G. F., Shen, R., Perozo, E. 2020; 583 (7814): 145-149

  Abstract

  Voltage-gated potassium (Kv) channels coordinate electrical signalling and control cell volume by gating in response to membrane depolarization or hyperpolarization. However, although voltage-sensing domains transduce transmembrane electric field changes by a common mechanism involving the outward or inward translocation of gating charges1-3, the general determinants of channel gating polarity remain poorly understood4. Here we suggest a molecular mechanism for electromechanical coupling and gating polarity in non-domain-swapped Kv channels on the basis of the cryo-electron microscopy structure of KAT1, the hyperpolarization-activated Kv channel from Arabidopsis thaliana. KAT1 displays a depolarized voltage sensor, which interacts with a closed pore domain directly via two interfaces and indirectly via an intercalated phospholipid. Functional evaluation of KAT1 structure-guided mutants at the sensor-pore interfaces suggests a mechanism in which direct interaction between the sensor and the C-linker hairpin in the adjacent pore subunit is the primary determinant of gating polarity. We suggest that an inward motion of the S4 sensor helix of approximately 5-7 Å can underlie a direct-coupling mechanism, driving a conformational reorientation of the C-linker and ultimately opening the activation gate formed by the S6 intracellular bundle. This direct-coupling mechanism contrasts with allosteric mechanisms proposed for hyperpolarization-activated cyclic nucleotide-gated channels5, and may represent an unexpected link between depolarization- and hyperpolarization-activated channels.

  View details for DOI 10.1038/s41586-020-2335-4

  View details for PubMedID 32461693

  View details for PubMedCentralID PMC7747229

• Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination. Journal of molecular biology Bailey, L. J., Sheehy, K. M., Dominik, P. K., Liang, W. G., Rui, H., Clark, M., Jaskolowski, M., Kim, Y., Deneka, D., Tang, W. J., Kossiakoff, A. A. 2018; 430 (3): 337-347

  Abstract

  Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones.

  View details for DOI 10.1016/j.jmb.2017.12.012

  View details for PubMedID 29273204

  View details for PubMedCentralID PMC5800945

• Solution NMR Studies of an Alternative Mode of Sin3 Engagement by the Sds3 Subunit in the Histone Deacetylase-Associated Sin3L/Rpd3L Corepressor Complex. Journal of molecular biology Clark, M. D., Zhang, Y., Radhakrishnan, I. 2015; 427 (24): 3817-23

  Abstract

  The Sds3 transcriptional corepressor facilitates the assembly of the 1- to 2-MDa histone deacetylase-associated Sin3L/Rpd3L complex by providing a crucial homodimerization activity. Sds3 engages the scaffolding protein Sin3A, via a bipartite motif within the Sin3 interaction domain (SID) comprising a helix and an extended segment. Here, we show that the SID samples two discrete, substantially populated conformations with lifetimes in the tens of milliseconds range. The two conformations differ via a translation of the main chain and the corresponding side chains in the 5- to 7-Å range. Given the close proximity of the SID to other functional motifs in Sds3 at the sequence level, the conformational exchange has the potential to regulate these activities.

  View details for DOI 10.1016/j.jmb.2015.10.018

  View details for PubMedID 26522936

  View details for PubMedCentralID PMC5084087

• Molecular Basis for the Mechanism of Constitutive CBP/p300 Coactivator Recruitment by CRTC1-MAML2 and Its Implications in cAMP Signaling. Biochemistry Clark, M. D., Kumar, G. S., Marcum, R., Luo, Q., Zhang, Y., Radhakrishnan, I. 2015; 54 (35): 5439-46

  Abstract

  The cyclic AMP response element-binding protein (CREB) is a signal-dependent transcription factor that exerts its positive effects on gene transcription of a broad range of genes by recruiting coactivators, including CREB-binding protein (CBP), its paralog, p300, and the family of CRTC (CREB-regulated transcriptional coactivators) proteins. Whereas recruitment of CBP/p300 is dependent on CREB phosphorylation at Ser133, recruitment of CRTCs is not. Here we describe how both mechanisms could concurrently drive transcription of CREB targets in a subset of head and neck cancers featuring chromosomal translocations that fuse portions of CRTC1 and CRTC3 genes with that of the Mastermind-like transcriptional coactivator MAML2. We show that a peptide derived from transactivation domain 1 (TAD1) of MAML2 binds to the CBP KIX domain with micromolar affinity. An ∼20-residue segment within this peptide, conserved in MAML2 orthologs and paralogs, binds directly to a KIX surface previously shown to bind to MLL1. The 20-residue MAML2 segment shares sequence similarity with MLL1, especially at those positions in direct contact with KIX, and like MLL1, the segment is characterized by the presence of an ∼10-residue helix. Because CRTC1/3-MAML2 fusion proteins are constitutively nuclear, like CREB, our results suggest constitutive recruitment of CBP/p300 to CREB targets that could be further enhanced by signals that cause CREB Ser133 phosphorylation.

  View details for DOI 10.1021/acs.biochem.5b00332

  View details for PubMedID 26274502

  View details for PubMedCentralID PMC4564341

• Structural insights into the assembly of the histone deacetylase-associated Sin3L/Rpd3L corepressor complex. Proceedings of the National Academy of Sciences of the United States of America Clark, M. D., Marcum, R., Graveline, R., Chan, C. W., Xie, T., Chen, Z., Ding, Y., Zhang, Y., Mondragón, A., David, G., Radhakrishnan, I. 2015; 112 (28): E3669-78

  Abstract

  Acetylation is correlated with chromatin decondensation and transcriptional activation, but its regulation by histone deacetylase (HDAC)-bearing corepressor complexes is poorly understood. Here, we describe the mechanism of assembly of the mammalian Sin3L/Rpd3L complex facilitated by Sds3, a conserved subunit deemed critical for proper assembly. Sds3 engages a globular, helical region of the HDAC interaction domain (HID) of the scaffolding protein Sin3A through a bipartite motif comprising a helix and an adjacent extended segment. Sds3 dimerizes through not only one of the predicted coiled-coil motifs but also, the segment preceding it, forming an ∼ 150-Å-long antiparallel dimer. Contrary to previous findings in yeast, Sin3A rather than Sds3 functions in recruiting HDAC1 into the complex by engaging the latter through a highly conserved segment adjacent to the helical HID subdomain. In the resulting model for the ternary complex, the two copies of the HDACs are situated distally and dynamically because of a natively unstructured linker connecting the dimerization domain and the Sin3A interaction domain of Sds3; these features contrast with the static organization described previously for the NuRD (nucleosome remodeling and deacetylase) complex. The Sds3 linker features several conserved basic residues that could potentially maintain the complex on chromatin by nonspecific interactions with DNA after initial recruitment by sequence-specific DNA-binding repressors.

  View details for DOI 10.1073/pnas.1504021112

  View details for PubMedID 26124119

  View details for PubMedCentralID PMC4507224