Hendrickson Laboratory

Wayne A. Hendrickson, Columbia University

 






Research in the Hendrickson Laboratory

Membrane
Receptors
& Signaling
Viral
Proteins
& HIV
Molecular
Chaperones
& Folding
Membrane
Proteins
& Genomics
Diffraction
Methods
& Synchrotrons
Biophysical
Principles

Most research projects of the Hendrickson laboratory aim for an atomic-level understanding of the biochemical action of biological macromolecules. This work entails the production and characterization of proteins and protein complexes from systems of interest, analysis of relevant structures by x-ray crystallography or cryogenic electron microscopy, and structure-inspired biochemical and computational studies of biochemical mechanisms and biophysical properties. Additionally, a continuing major emphasis is in the relevant methodology development.

      Current activities emphasize membrane receptors as related to transmembrane signal transduction, viral proteins especially as related to HIV infection, molecular chaperones as related to protein folding and aggregation, membrane proteins as studied through structural genomics, diffraction methodology and synchrotron instrumentation, and investigations on biophysical principles.

      Although our research is at a very basic level, many of the studies have biomedical relevance. Notably, our work on HIV gp120 relates quite immediately to vaccine and drug development, our studies on the FSH receptor system relate directly to reproductive biology, and our work on Hsp70 chaperones is relevant to cancer and to neurodegenerative diseases, and our work on bestrophin ion channels relates directly to eye disorders.


Membrane Receptors and Cellular Signaling   

An important emphasis of our research concerns the initial phases of cellular signal transduction, including the biochemical and biophysical aspects of signal transduction across membranes by major signaling systems (Hendrickson, 2005). In most cases, the signal-initiating stimulus from the environment is chemical; it may be a small compound, a macromolecular hormone or growth factor, or even another cell. Nearly always, receptors embedded in the cellular membrane mediate transmission of signaling into the cell. Our interest lies in the mechanisms by which biochemical signals are transduced across the membrane. We currently concentrate on complete integral membrane receptor proteins, but relevant extra-membranous portions are also studied. We previously contributed to understanding of receptor tyrosine kinases with We have current efforts on several receptor classes:

Receptor tyrosine kinases.We previously contributed to understanding of tyrosine kinases with the first structures of tyrosine kinase domains (insulin-receptor kinase: Hubbard et al., 1994; lymphocyte kinase: Yamaguchi & Hendrickson, 1996), with structures of extracellular portions of the T-cell co-receptor CD4 (Ryu et al., 1990; Wu et al., 1997), with the structure of fibroblast growth factor (FGF) complexed with an FGF receptor ectodomain (Stauber et al., 2000), and with structures of several tyrosine kinase ligands [CNTF: (McDonald et al., 1995); FGF: (DiGabrielle et al., 1998); SCF (Jiang et al., 2000)].

Histidine kinase receptors.Typical histidine kinase receptors have a sensor domain separated by two transmembrane helices and followed by a cytoplasmic portion that contains the kinase domain and a histidine autophosphorylation site. The sensor domains are highly variable, specific to different ligands; whereas the cytoplasmic portions are more conserved, especially so for the kinase domains. Using a structural genomics approach, we systematically pursued representative structures for sensor domain families that we have characterized. Several sensor structures have been solved (PhoQ: Cheung et al., 2008; DcuS and DctB: Cheung & Hendrickson, 2008; NarX: Cheung & Hendrickson, 2009; HK29: Cheung et al., 2009; TorS: Moore & Hendrickson, 2009; HK1: Zhang & Hendrickson, 2010; TorT/TorS: Moore & Hendrickson, 2012; HK3: Zhang & Hendrickson, 2014), and others are in progress. We have also completed structures on cytoplasmic domains, including one of the entire cytoplasmic dimer (Marina et al., 2005), comprising both its coiled-coil dimerization domain and its kinase domains. Taken together, these structural results are inspiring testable hypotheses about mechanisms for signal transduction in this system. Our current focus is on producing active, intact histidine kinase receptors for crystallographic analysis.

G-protein coupled receptors (GPCRs). We commit a substantial effort toward understanding the conformational states of the seven-transmembrane (7TM) receptors that function through stimulation of guanine nucleotide exchange in the Gα subunit of a heterotrimeric G protein. The major focus is on serotonin receptors and on glycoprotein hormone receptors, but we also work on other GPCRs including the chemokine receptors involved in HIV infection. Much of the effort to date has focused on producing intact molecules for crystallization, including the characterization of dimers (Mancia et al., 2008). This effort entails complexes with antibodies, which we can prepare (Mancia et al., 2007) and produce as recombinant Fab particles (Assur et al., 2007). We determined the crystal structure of the complex between human follicle stimulating hormone (FSH) and the hormone-binding portion of the human FSH receptor (FSHRHB) (Fan & Hendrickson, 2005), and we are now producing full-length LH and FSH receptor in complexes with their respective glycoprotein hormones.

Ion channel receptors. In parallel with our work on GPCRs, we also study two types of ligand-gated ion channels. The first of these are Cys-loop ion channels where we have studied four ligands bound to the extracellular domain of the chicken &alpha9 9 acetylcholine receptor. These structures provide important insights into conformational changes that mediate ligand gating of the ion channel opening. The second system of interest concerns the ryanodine-sensitive calcium-release channel known as the ryanodine receptor. Working in collaboration with the laboratory of Andrew Marks, we have obtained nicely diffracting crystals of the intact 2.3MDa receptor and working with the Marks and with Joachim Frank laboratories we have recently obtained cryo-EM structures that could be interpreted with a C&alpha9 trace for the entire ordered portion of the polypeptide chain (Zalk et al., 2015). Work is in progress on open-state structures.



Viral Proteins and HIV Infection   

HIV envelope interactions. The foundation of our work on interactions of the HIV envelope proteins with cellular receptors lies in structures of complexes between HIV gp120 and both its the cellular receptor CD4 and a neutralizing antibody bound to the co-receptor binding site. These were determined both for a laboratory adapted R4 strain, HxBc2 (Kwong et al., 1998), and for a primary R5 isolate, Yu2 (Kwong et al., 2000), and in each case CD4 was represented by the D1D2 binding fragment and the antibody component was the human 17b Fab fragment. We had previously determined structures for soluble CD4 (Ryu et al., 1990; Wu et al., 1997). We subsequently carried out studies on the thermodynamics of these interactions (Myszka et al., 2000; Kwong et al., 2002), and we have determined a number of additional structures including complexes with CD4 mimetics (Huang et al., 2005). Recent work focuses on the development of antagonists of the gp120-CD4 interaction. Toward this end, we designed derivatives of F43C CD4 (D1D2) in which cysteine adducts bind into the Phe43 interfacial cavity (Xie et al., 2007), and we have determined four structures of such complexes. all in the HxBc2 lattice. These structures provide details of interactions that are being used to design and synthesize new compounds with the ultimate aim of obtaining useful HIV entry inhibitors. More recently, we have determined structure of small-molecule entry inhibitors and are using structure-based design methods to develop these compouds (LaLonde et al., 2013).

Other viral proteins.We also have programs directed at other viral proteins, including other HIV envelope receptors. In particular, we have analyzed structures of the dendritic-cell receptor DC-SIGN, a tetrameric C-type lectin that attaches to sites of glycosylation on HIV gp120 and also on Dengue virus. In collaboration with Michael Rossmann, we earlier showed how the lectin domain binds to Dengue virus (Pokidysheva et al., 2006) and now with the tetrameric stalk model in hand we provide further definition. Finally, in collaboration with Steve Goff, we have determined two crystal structures of the capsid protein from Moloney murine leukemia virus (MMLV), a retrovirus analogous with HIV, that correspond respectively to the hexagonal lattices of the immature and the mature viral capsid.



Molecular Chaperones and Protein Folding   

Hsp70 chaperones.The 70kD family of heat shock protein (Hsp70) chaperones is ubiquitous, having involvement in diverse activities in all organisms. Others had previously characterized the ATPase domain of Hsp70s and we determined the first structure of an Hsp70 substrate-binding domain, that of DnaK as associated with a high-affinity peptide (Zhu et al., 1996). The nature of the allosteric interaction between the ATPase substrate-binding units in the chaperone cycle of bindings and releases has long remained elusive, however. Our structure of yeast Sse1 (Liu & Hendrickson, 2007), an Hsp110 family member and a clear relative of Hsp70s based on its structure, provided the first clear picture for these interactions. The structure showed a remarkable change in conformation relative to that in domains from Hsp70s. A battery of interface mutations in Sse1 and its DnaK homolog, tested in yeast and bacteria, respectively, informed us about general modes of conformational change and ATPase action. In vitro biochemical tests of several of the DnaK mutants have inspired a new model for the chaperone cycle and the generation of mutant-stabilized ATP states that have succumbed to crystallization. In collaboration with Qinglian Liu, we have determined the structure of DnaK in an Sse1-like restraining state as complexed with ATP (Qi et al., 2013), and we are at work on what we characterize to be the Hsp70 stimulating state.

Other molecular chaperones.Molecular chaperones play vital roles in protein folding, in the suppression of aggregation in states of cellular stress, and specialized roles in protein trafficking. We have made substantial progress in the biochemical and structural characterization of four classes of molecular chaperones. Besides the Hsp70s, these include the important bacterial chaperone trigger factor (Martinez-Hackert & Hendrickson, 2009) and an FKBP-based chaperone from Methanococcus (Martinez-Hackert & Hendrickson, 2011). Our fourth class of interest is in Boca/MESD, a specialized chaperone that mediates the folding of LDL receptors and related proteins. We have analyzed crystal structures of the Boca/MESD chaperones from three species (Collins & Hendrickson, 2011). In addition, we have analyzed the role played by coiled-coil interactions in the aggregations associated with protein folding disorders (Fiumara et al., 2010).



Membrane Proteins and Structural Genomics   

We are among the principal participants in the New York Consortium on Membrane Protein Structure (NYCOMPS), which has been supported by the NIGMS Protein Structure Initiative (PSI). Together with our NYCOMPS colleagues, we created an efficient pipeline for the expression and production of membrane proteins (Punta et al., 2009; Love et al., 2010). NYCOMPS has become highly productive in the support of studies of membrane proteins strategically selected to illuminate the universe of membrane proteins and in supporting projects nominated by the community at large. We are involved in developing the technology for protein production and for structure determinations (Liu et al., 2012), and we are particularly engaged in the structure determination component of the project. We have brought several NYCOMPS proteins into our laboratory for crystallization and structural analysis, which in each case we have characterized in functional assay both for the bacterial protein for which we obtained stuctures and for better studied eukaryotic homologs.

        The first of the NYCOMPS structure solved by my group was that of bacterial protein TehA. This protein proved to be a homolog of Slow Anion Channel 1 (SLAC1), which controls stomatal closure in plant leaves in response to darkness and to environmental factors such as drought and high CO2 levels. We determined TehA structures with extraordinary detail (down to 1.15Å resolution), and we characterized the channel activity of both TehA and Arabidopsis SLAC1 (Chen et al., 2010). More recently, working in collaboration with Qun Liu, we determined the structure of a bacterial homolog of human Bax-inhibitor 1 (BI-1) and performed informative functional characterizations (Chang et al., 2014). We also recently determined the structure of another bacterial channel protein, which is the counterpart of human bestrophin 1 known for its association with early-onset macular degeneration. Here again we analyzed channel activities for both homologs (Yang et al., 2014). Most recently, we have completed an analysis of tryptophan-rich sensory proteins (TSPOs) from a bacterium for initial structures and from vertebrates for functional characterization. We tested structure-inspired hypotheses to establish a previously unappreciated role of TSPO proteins in degrading porphyrins for the control of reactive oxygen species (Guo et al., 2015). A number of other NYCOMPS structures have been solved and characterizations and analyses are in progress.



Diffraction Methods and Synchrotron Radiation

Our laboratory has been engaged in the development of methods for diffraction analysis of biological structure for a long time. Early contributions include widely used phasing coefficients (Hendrickson & Lattman, 1970), the initiation of stereochemically restrained refinement of crystal structures (Hendrickson & Konnert, 1980; Konnert & Hendrickson, 1980), and the structural analysis of crambin based solely on anomalous scattering from sulfur atoms (Hendrickson & Teeter, 1981). The crambin structural analysis established what is now known as the single-wavelength anomalous diffraction (SAD) method and paved the way for his development of the multi-wavelength anomalous diffraction (MAD) method (Hendrickson, 1985; Hendrickson et al., 1988). Broad utility of the MAD method followed when we recognized that selenium could serve as a rich source for the required diffraction signals (Hendrickson et al., 1989) and that selenomethionine (SeMet) could be substituted readily for the natural amino acid methionine (Hendrickson et al., 1990; Yang et al., 1990). We tested MAD phasing in applications at synchrotrons around the world, and with HHMI support we developed NSLS beamline X4A at Brookhaven National Laboratory to optimize the MAD experiment (Staudenmann et al., 1989). X4A became highly productive and MAD beamlines were emulated around the world. MAD and SAD methods now dominate in biological crystallography, producing many hundreds of new structures each year (Hendrickson, 2014). Recently, we have advanced methods for SAD phasing analysis of native macromolecules, using low x-ray energy to enhance anomalous signals and multiple crystals to reduce noise (Liu et al., 2012; Liu et al., 2013; Liu et al., 2014).

        We honed anomalous scattering methods in applications to tough problems at the forefront of biology and medicine. Among these are the following: (1) MAD phasing was used to determine the initial structure of the human CD4 (Ryu et al., 1990) and SeMet CD4 produced in mammalian cells was used to determine the full-length structure (Wu et al., 1997). CD4 is a T-cell co-receptor for the cellular immune response, but it is also used by the AIDS virus to initiate infection and later structures were determined in complexes with the HIV envelope glycoprotein gp120. These studies revealed atomic details that are providing insights for vaccine development and inhibitor design. (2) MAD phasing was also used to determine the first protein tyrosine kinase structures, those of the human insulin receptor (Hubbard et al., 1994) and of human lymphocyte kinase Lck (Yamaguchi & Hendrickson, 1996). Besides generating deep insights into diabetes and the cellular immune response, these analyses also paved the way for a new generation of cancer therapeutics. (3) Another early application was to the reproductive hormone human chorionic gonatoropin (hCG) which was determined by SeMet MAD phasing (Wu et al., 1994). Another reproductive hormone, human follicle-stimulating hormone (FSH) was later analyzed in complex with its receptor (Fan et al., 2005). FSH is used therapeutically to treat infertility, and the hormone-receptor complex provided lead concepts both for enhancing fertility and for contraception. (4) The structures of Hsp70 molecular chaperones determined by SeMet MAD phasing (Zhu et al., 1996; Liu & Hendrickson, 2007) have provided mechanistic insights of relevance for activities that protect against neurodegenerative diseases such as Alzheimer's and Parkinson's. (5) Recently, we have determined several membrane-protein structures using SAD phasing (Chen et al., 2010; Chang et al., 2014; Yang et al., 2014; Guo et al., 2015). These studies on membrane proteins are providing novel mechanistic insights relevance ranging from environmental concerns to eye disorders.

        Besides the applications in these thematic areas, we have also advanced MAD and SAD phasing methods in applications to dozens of other structural problems. These include myohemerythrin (Sheriff et al., 1987), streptavidin (Hendrickson et al., 1989), clam hemoglobin (Royer et al., 1989), ribonuclease H (Yang et al., 1990), a C-type lectin (Weis et al., 1991), fibronectin (Leahy et al., 1992), reverse transcriptase (Georgiadis et al., 1995), N-cadherin (Shapiro et al., 1995), biotin carboxyl carrier protein (Athappilly & Hendrickson, 1995), UmuD SOS protein (Peat et al., 1996), myelin p0 (Shapiro et al., 1996), HIT proteins (Lima et al., 1996; Lima et al., 1997), hemocyanin (Cuff et al., 1998), α -tocopherol transfer protein (Min et al., 2003), Notch CSL-DNA (Kovall & Hendrickson, 2004), mitochondrial SCO1 (Williams et al., 2005), and dynein TcTex-1 (Williams et al., 2005). These studies have provided significant insight into mechanisms and principles, often as related to human biology.

        Synchrotron radiation is essential for effective MAD experiments and it greatly enhances SAD experiments. We developed the X4 beamlines at the National Synchrotron Light Source (NSLS), now supported by the New York Structural Biology Center (NYSBC), to test phasing methods, and this work continues with enhancements in x-ray optics (Lidestri & Hendrickson, 2007) and in applications. We are now well advanced in constructing the NYSBC Microdiffraction Beamline NYX at NSLS-II. This new beamline will exploit a novel design to provide exceptional energy resolution to optimize anomalous signals. Working with the ADSC detector company, we have co-developed a pixel array detector for NYX, which is being tested at an NE-CAT beamline at the Advanced Photon Source (APS) at Argonne ahead of its deployment at NYX. NYX is being developed in anticipation of accommodation for approved companion beamline LAX, which will be specialized for Low-energy Anomalous X-ray diffraction experiments. We have also recently conducted experiments at the Linac Coherent Light Source, Stanford's x-ray free electron laser (FEL). We are continuously engaged in optimizing instrumentation and methodological approaches for exploiting synchrotron radiation in biological crystallography, especially with respect to MAD and SAD experiments.



Biophysical Principles

We have longstanding interests in studies on the biophysical principles of protein structure, dynamics and evolution. Based largely on our early analyses of restrained refinements of crystallographic B factors (Konnert & Hendrickson, 1980; Hendrickson & Konnert, 1980; Hendrickson, 1989), we performed several analyses on the extractions of information about protein dynamics from atomic mobility parameters (B factors). Examples include a molecular dynamics comparison with crystal structure (Yu et al., 1985), an analysis of epitope antigenicity (Tainer et al., 1984), a study of structural heterogeneity in crystallized proteins (Smith et al., 1986), and a study of disorder by x-ray restrained molecular dynamics (Kuriyan et al., 1991). Earlier, we contributed to understanding of fundamental processes in radiation damage (Hendrickson, 1976). We have also performed analyses on molecular evolution (Aronson et al., 1994; Shapiro et al., 1995) and in comparative analyses of molecular structure, both routinely in our structure descriptions and more generally across families (Fan & Hendrickson, 2008; Cheung & Hendrickson, 2010; Zhang et al., 2010). Most recently, we have developed a robust computational procedure for computing realistic pathways for large-scale conformational transitions (Korkut & Hendrickson, 2009a,b), which we have applied to conformational transforamtions in HIV envelope proteins (Korkut & Hendrickson, 2012) and to Hsp70 chaperones.




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