New York Structural Biology Discussion Group: 4th Winter Meeting

The New York Structural Biology Discussion Group brings together tri-state structural biologists, biophysicists, and biochemists to discuss findings in an informal setting thorough talks and poster sessions. Each bi-annual meeting includes presentations by graduate students, post-docs and laboratory heads. Talks are selected from area laboratories by the program committee with an emphasis on new and emerging data and techniques. There will be an evening reception after the meeting has concluded.

Organizers: Hao Wu, Weill Medical College of Cornell University; David Stokes, NYU School of Medicine; David Eliezer, Weill Medical College of Cornell University

Speakers: Paramjit Arora, New York University; Ewa Folta-Stogniew, Yale University; David Gadsby, Rockefeller University; David F. Green, Stonybrook University; Ya Ha, Yale University; Steve Hubbard, New York University; Jin Kim Montclare, Polytechnic University; Crina Nimigean, Weill Medical College of Cornell University; Rajesh Kumar Prakash, Cold Spring Harbor Laboratory; David Provasi, Mount Sinai School of Medicine; Daniel Schmidt, Rockefeller University; Vinzenz Unger, Yale University

Agenda

Synthetic Approaches for Targeting Protein Interfaces
Paramjit Arora, New York University

Application of Light Scattering for Analysis of Protein Oligomerization and Protein-Protein Interactions
Ewa Folta-Stogniew, Yale University

The Ion Pathway Through the Na,K-ATPase Pump
David Gadsby, Rockefeller University

The Structural Glycobiology of HIV Infection and Prevention: Insights from Computational Models
David F. Green, Stonybrook University

The Role of Membrane Thinning in Intramembrane Proteolysis
Ya Ha, Yale University

Structural Basis for Protein Recruitment to the Insulin Receptor and Muscle-Specific Kinase
Steve Hubbard, New York University

Structural and Functional Insight into A. oryzae Cutinase: Biocatalyst for Synthetic Ester and Polyester Degradation
Jin Kim Montclare, Polytechnic University

A Novel Mechanism of Potassium Channel Selectivity Revealed by Ionic Interactions within the Pore
Crina Nimigean, Weill Medical College of Cornell University

Turning on Transcription : A Sugary Tale Revisited
Rajesh Kumar Prakash, Cold Spring Harbor Laboratory

Simulating Long Timescale Biological Processes with Metadynamics
David Provasi, Mount Sinai School of Medicine

Tuning of Kv Channel Function and Pharmacology by the Mechanical State of the Lipid Membrane
Daniel Schmidt, The Rockefeller University

Insights into the Mechanism of Copper Transport
Vinzenz Unger, Yale University

Abstracts

Synthetic Approaches for Targeting Protein Interfaces
Paramjit Arora, New York University

Proteins often utilize small folded domains for recognition of other biomolecules. The hypothesis guiding our research efforts is that by mimicking these folded domains, we can reproduce the function of a particular protein with metabolically stable synthetic molecules. We are pursuing various approaches that provide peptidic and nonpeptidic scaffolds displaying protein-like functionality with sequence- specificity. This talk will primarily discuss structure and function in hydrogen bond surrogate based artificial helices.

Application of Light Scattering for Analysis of Protein Oligomerization and Protein-Protein Interactions
Ewa Folta-Stogniew, Ph.D., Yale University

Size-exclusion chromatography (SEC) coupled with "on-line" static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection provides a universal approach for determination of the molar mass and oligomeric state in solution of native proteins as well as glycosylated proteins or membrane proteins solubilized in non-ionic detergents. In the SEC-UV/LS/RI approach, SEC serves solely as a fractionation step while the responses from the three detectors are utilized to calculate the molar mass for the polypeptide portion of the native or modified protein. The amount of sugar, or lipid, or detergent bound to the polypeptide chain can also be estimated from the SEC-UV/LS/RI analysis. This presentation discusses capabilities and limitations of light scattering applications with emphasis on detection and quantitation of protein homo- and hetero-oligomers, including complexes formed by membrane proteins solubilized in detergent's micelles and protein-nucleic acid complexes.

The Role of Membrane Thinning in Intramembrane Proteolysis
Ya Ha, Yale University

Intramembrane proteolysis is important for many cell functions. This reaction is catalyzed by highly specialized integral membrane proteases. Here I discuss possible mechanisms of intramembrane proteolysis, drawing lessons from the recently solved crystal structures of the membrane protease. The role of membrane thinning around the protease, and how it may facilitate the unfolding of transmembrane alpha-helices, will also be discussed.

Structural Basis for Protein Recruitment to the Insulin Receptor and Muscle-Specific Kinase
Steve Hubbard, New York University

The insulin receptor and MuSK (muscle-specific kinase) are members of the receptor tyrosine kinase family of cell-surface receptors. Upon activation by their respective ligands (insulin and agrin), these receptors undergo trans-phosphorylation on specific tyrosines in their cytoplasmic domains, which serve as docking sites for downstream signaling proteins. The adapter protein Grb14 is a negative regulator of insulin signaling and consists of a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, an inhibitory BPS (between PH and SH2) region, and a C-terminal Src-homology-2 (SH2) domain. A recent crystal structure of the RA and PH domains of Grb10 (highly related to Grb14) has revealed that these two domains are structurally coupled, which in Grb14 serves to integrate Ras and membrane-phosphoinositide binding. Dok7 is an essential adapter protein in formation of the neuromuscular junction, and is recruited to activated MuSK via its PH and phosphotyrosine-binding (PTB) domains. Our crystal structure of the tandem PH and PTB domains of Dok7 have revealed how Dok7 is selectively recruited to MuSK to stimulate MuSK's catalytic (tyrosine kinase) activity.

Structural and Functional Insight into A. oryzae Cutinase: Biocatalyst for Synthetic Ester and Polyester Degradation
Jin Kim Montclare, Ph.D., Polytechnic University

The Apergillus oryzae cutinase is responsible for hydrolysis of the protective cutin lipid polyester matrix in plants. Here we have determined a 1.8 Å resolution crystal structure of A. oryzae cutinase and compared it to the well-studied enzyme from Fusarium solani. Although the overall structure is similar to that of F. solani, with the same folds, catalytic triad and regions of flexibility, there are three critical differences in which the A. oryzae structure displays: (i) a larger catalytic pocket; (ii) an additional disulfide bond; and (iii) a more hydrophobic surface area near the active site. These structural features of A. oryzae cutinase result in improved overall hydrolytic activity, selectivity for long chain substrates, reactivity towards synthetic polyesters and overall stability. The structure-function relationship presented here establishes a paradigm for understanding other enzymes that can hydrolyze synthetic esters or polyesters and insight into engineering new cutinase-inspired biocatalysts with tailor- made properties.

A Novel Mechanism of Potassium Channel Selectivity Revealed by Ionic Interactions within the Pore
Crina Nimigean, Weill Medical College of Cornell University

Potassium channels allow K+ ions to easily diffuse through their pores while effectively preventing the smaller Na+ ions from permeation. This selection process occurs at the narrow selectivity filter in the pore. The current hypothesis for the mechanism of K+ channel selectivity against Na+ ions is that K+ binding in the filter is favorable, while Na+ binding in the filter is an energetically unfavorable process. Using KcsA as a model system, we were able to propose a fundamentally different mechanism for selectivity against smaller ions such as Na+ and Li+. Using single channel blocking experiments, X-ray crystallography, and molecular dynamics simulations, we showed that Na+ and Li+ ions can bind in the selectivity filter with high affinity, albeit at a different site than K+. Intracellular Na+ and Li+ ions are selected against before they enter the filter due to a large energy barrier associated with moving K+ ions upwards inside the filter when a Na+ or Li+ ion is in the cavity.

Turning on Transcription : A Sugary Tale Revisited
Rajesh Kumar Prakash, Cold Spring Harbor Laboratory

Saccharomyces cerevisiae can utilize a variety of carbon sources. Galactose is one such carbon source, which enters the cytoplasm through a permease. This Galactose is sequentially converted to pyruvate through several steps by the action of GAL enzymes and pyruvate eventually enters the Krebs cycle to produce energy. Transcriptional regulation of these galactose metabolizing genes in Saccharomyces cerevisiae is tightly regulated by three core proteins - Gal4p, the transcriptional activator that binds to upstream activating DNA sequences (UASGAL), Gal80p, a repressor that binds to the C-terminus of Gal4p and inhibits transcription, and Gal3p, a cytoplasmic transducer which upon binding the in-coming galactose in an ATP dependant manner, relieves Gal80p repression. The current model of induction relies on Gal3p sequestering Gal80p in the cytoplasm and hence implies a nucleo-cytoplasmic shuttling role for Gal80p. However, the rapid induction (only a couple of minutes) of this system implies that there is a missing factor. Our structure of Gal80p in complex with a peptide from the C-terminal activation domain of Gal4p reveals the existence of a dinucleotide co-factor that mediates the interaction between the two. Biochemical and in vivo experiments suggest that NADP plays a key role in the initial induction event by dislodging Gal80p from the Gal4p activation domain and hence initiates transcription of the GAL genes.

Tuning of Kv Channel Function and Pharmacology by the Mechanical State of the Lipid Membrane
Daniel Schmidt, The Rockefeller University

Voltage-dependent K+ (Kv) channels underlie action potentials through gating conformational changes that are driven by membrane voltage. In a recent study of the paddle chimera Kv channel we could demonstrate that the rate of channel opening, the voltage-dependence of the open probability, and the maximum achievable open probability depend on the lipid membrane environment. The activity of the voltage sensor toxin VsTx1, which interferes with voltage-dependent gating by partitioning into the membrane and binding to the channel, is also dependent on the membrane. Membrane environmental factors that influence channel function are divisible into two general categories: lipid compositional and mechanical state. The mechanical state can have a surprisingly large effect on the function of a voltage-dependent K+ channel, including its pharmacological interaction with voltage sensor toxins. The dependence of VSTx1 activity on the mechanical state of the membrane lead us to hypothesize that voltage sensor toxins exert their effect by perturbing the interaction forces that exist between the channel and the membrane.

Insights into the Mechanism of Copper Transport
Vinzenz Unger, Yale University

Copper uptake proteins (CTRs), mediate cellular acquisition of the essential metal copper in all eukaryotes. Using electron cryomicroscopy, we determined a 3D-structure of hCTR1 at ~7Å resolution. The structure suggests that CTR1 proteins transport copper through a movement of Cu(I)-ions between defined binding sites and that intracellular copper chaperones are capable of directly obtaining copper from CTR1. To test these ideas, we used EXAFS to determine copper binding sites in hCTR1. We find that trimeric hCTR1 can stably bind 2 Cu(I)-ions through 3-coordinate Cu-S bonds. Moreover, EXAFS data obtained using Se-Cys-labeled CCS is consistent with the idea that the chaperone can obtain Cu(I) directly from copper loaded hCTR1. Modeling of a hypothetical hCTR1-CCS complex furthermore suggests that CCS may be able to associate with the membrane. Such a partitioning would greatly increase the efficiency of copper transfer to the chaperone because it would allow CCS to "find" hCTR1 through a 2D-diffusional search rather than a 3D-random walk. In support of this idea, we find that hCCS can bind to bilayers in vitro. Lastly, we generated a C-alpha model of the membrane embedded region of hCTR1 to aid future mechanistic studies. The model is consistent with the results of an extensive Trp-scan analysis of the membrane domain of hCTR1 and yCTR3 in that the overwhelming majority of residues found to be structurally and/or functionally important participate in helix packing interactions or face the copper permeation pathway along the 3-fold axis the CTR trimer.

Sponsors:

• The NSF Research Coordination Network
• GE Healthcare
• Nature Structural & Molecular Biology
• TTP Labtech
• Rigaku
• FEI Company
• Imclone Systems
• JEOL
• Varian
• Roche Pharmaceuticals