14^th International Conference on Structural Biology September 24-26, 2018 Berlin, Germany

Chair

Joachim Krebs

MPI for Biophysical Chemistry, Germany

Session Introduction

Peter B Stathopulos

University of Western Ontario, Canada

Title: Structural elements of stromal interaction molecule mediated store operated calcium entry regulation

Biography:

Peter B Stathopulos applies structural biology approaches to reveal molecular mechanisms driving calcium signaling processes in health and diseased states including

heart disease, cancer and immunodefi ciency. He integrates nuclear magnetic resonance spectroscopy and x-ray crystallography with a host of biophysical, chemical

biology and live cell methodologies to understand the relationship between structure and function of critical calcium signaling proteins. Ultimately, this structure-function

data is used for the rational identifi cation of new drug binding targets with the potential to modulate these pathways to maintain health or treat disease.

Abstract:

Calcium (Ca2+) is a universal signaling entity in eukaryotic cells mediating diverse processes such as the immune response,

hypertrophy, apoptosis, platelet aggregation and memory, to name a few. Th ese processes require a sustained elevation

of cytosolic Ca2+ levels which is facilitated by store operated Ca2+ entry (SOCE). SOCE is the process whereby endoplasmic

reticulum (ER) luminal Ca2+ depletion signals the opening of ion channels on the plasma membrane (PM) which facilitate

the movement of Ca2+ down the concentration gradient from the extracellular space into the cytosol. Th e principal molecules

that mediate SOCE include the ER resident stromal interaction molecule-1 (STIM1) and PM ORAI1 protein subunits which

assemble into a channel pore. Upon ER luminal Ca2+ depletion, STIM1 undergoes a destabilization coupled oligomerization

which leads to translocation of this Ca2+ sensor to ER-PM junctions where it couples to ORAI1 subunits and opens these PM

Ca2+ channels. Since the identifi cation of STIM1 and ORAI1 as the principal molecules driving SOCE, considerable progress

has been made elucidating their high-resolution structural mechanisms of action. Author will present available structural data

on the STIM1 Ca2+ sensing mechanism and how this regulator may complex to ORAI1 subunits. Th e coupling mechanism

revealed using soluble human STIM1 and ORAI1 fragments are congruent with the hexameric assembly elucidated in the D.

melanogaster crystal structure. Finally, author will present unpublished work showing how post translational modifi cations

within the luminal domain of STIM1 aff ects the structural mechanisms of Ca2+ sensing. Ultimately, the post-translation

modifi cation driven STIM1 structural and biophysical changes have implications in the agonist induced hypertrophic response

and pinpoint a new therapeutic target for heart disease.

Toshiya Senda

High Energy Accelerator Research Organization (KEK), Japan

Title: Automated systems for X-ray crystallography at Photon Factory

Biography:

Toshiya Senda has completed his PhD from Nagaoka University of Technology, Niigata, Japan in 1995. He was a Research Associate in Nagaoka University of Technology

during 1995-2001 and a Senior Researcher in Institute of Advanced Industrial Science and Technology during 2001-2012. Now, he is the Director/Professor of Structural

Biology Research Center of High Energy Accelerator Research Organization (KEK) in Japan. He was awarded the CrSJ (Crystallographic Society of Japan) award in 2014.

Abstract:

X-ray crystallography has been the most widely utilized method for 3D structure determination of biological macromolecules

at atomic resolution. Since modern biology is studied at the molecular level, the 3D structures of biological macromolecules

provide critical information for rational biological studies. X-ray crystallography is therefore becoming an essential tool for

molecular and cellular biology and is increasingly being adopted by non-crystallographers. To facilitate this movement, the

Structural Biology Research Center (SBRC) in KEK has developed several robotics and automation systems for crystallization,

data collection, and structure determination. For the initial crystallization screening, we have developed protein crystallization

system (PXS) that can automatically perform initial crystallization screenings in a sitting drop setting. Th e PXS possesses

an automated image acquisition function for crystallization droplets. Th e obtained images can be checked from outside the

KEK campus via a VPN connection. Moreover, the crystallization plate can be directly mounted to a specifi c goniometer at

BL-17A for in situ data collection. Our protein crystallography beamlines, BL-1A, 5A, 17A, AR-NW12A, and AR-NE3A,

are highly automated by the PReMo program. Once a cassette containing protein crystals is set to the system, a robot can

mount a crystal to the goniometer in a beamline; do the centering of the crystal and start data collection. Beginning in May

2018, we will provide a fully automated data collection service for users. In this service, the obtained diff raction data sets will

be automatically processed by a data-processing routine in PReMo. Th e processed data can then further be analyzed with

PReMo for structural determination using the SAD method. Although at present the structure determination following the

data processing must be performed manually, we are developing a new routine for automated structure determination. It is

of note that all these automated functions are based on a database system in PReMo, and thus data from crystallization to

structure determination can be treated in an integrated manner. Our service is expected to provide the basis of an intelligent

system for crystallography.

Grace E Stutzmann

The Chicago Medical School - Rosalind Franklin University, USA

Title: Calcium signaling in the CNS in health and neurodegenerative disease

Biography:

Grace E Stutzmann focuses on early mechanisms of neurodegenerative disorders. She received her PhD from New York University, Center for Neural Science and

completed her Postdoctoral fellowships at Yale University and UC Irvine. She is currently an Associate Professor at Rosalind Franklin University/The Chicago Medical

School and serves as the Director for the Center for Neurodegenerative Disease and Therapeutics. Her research investigates effects of calcium signaling dysregulations

associated with AD on neuronal physiology, synaptic transmission, and histopathology. She uses transgenic mice expressing human mutations that cause familial AD and

employs techniques such as in vitro electrophysiology and 2-photon calcium imaging to study activity in neurons, in addition to immuno-based assays, molecular biology

and behavioral approaches. Her research has shown that specifi c calcium-mediated signaling pathways are dysregulated in AD and over time, facilitate amyloid plaques

and tangles formation, interfere with memory encoding circuits, and eventually can kill the cell.

Abstract:

Statement of the Problem: A shared aspect among many neurodegenerative disorders is a limited understanding of cellular

disease mechanisms. In our research, we have found that dysregulated calcium channels play a central and early role in driving

diseases such as Alzheimer’s, Huntington’s and brain injury. Specifi cally, defects in intracellular calcium stores within the

endoplasmic reticulum (ER) and their resident release channels such as the ryanodine receptor (RyR) are upstream of the

major disease features such as protein aggregates and synaptic defi cits. By focusing upon the proximal calcium channelopathy,

rather than later-stage features, signifi cant advances in understanding disease etiology may be made.

Methodology & Th eoretical Orientation: Whole cell patch clamp electrophysiology combined with 2-photon calcium

imaging are used to record signaling from hippocampal neurons in acute brain slices from mouse models of Alzheimer’s

disease (AD) and human neurons transformed from AD patients. Immunoassays and qRT PCR probe protein and transcript

levels in mouse and human neurons.

Findings: In early stage AD mice, RyR-evoked calcium signals are signifi cantly greater than non-transgenic controls, while

calcium signaling though voltage-gated channels and NMDA receptors are not diff erent. Similar observations were made

in human neurons from AD patients. Additionally, RyR 2 message is increased in both the mouse and human AD neurons.

Pharmacologically normalizing RyR-calcium release restores RyR 2 levels in mice, as well as reduces many of the AD features

including beta amyloid, hyperphosphorylated tau and synaptic loss.

Conclusion & Signifi cance: Dysregulated ER calcium channel functioning is an early feature of AD in mouse models and

human samples and likely initiates a pathogenic cascade in the proximal disease stages. Because of the multiple and essential

roles of calcium signaling in neurons, alterations in RyR-channel properties can generate multiple downstream maladaptive

eff ects such as those seen in neurodegenerative disorders.

Jan B Parys

KU Leuven, Belgium

Title: Functional properties of the IP3 receptor at membrane contact sites

Biography:

Jan B Parys is a faculty member since 1997, ordinary Professor of Physiology since 2006 and Head of the Laboratory of Molecular and Cellular Signaling at the KU

Leuven, Belgium since 2013. His research always concerned the understanding of the mechanisms for Ca2+ homeostasis in general and the structure, function and

regulation of the inositol 1, 4, 5-trisphosphate receptor (IP3R), a central player in the initiation and propagation of intracellular Ca2+ signals, in particular. More recently, he

focused on the role of the IP3R in conjunction with other Ca2+-transporting proteins of the endoplasmic reticulum, the mitochondria and the lysosomes in cell survival and

cell death, and especially in the processes of apoptosis and autophagy. Since 2010 he is Secretary-General of the European Calcium Society (ECS) and was organizer

or co-organizer of multiple international workshops (2013) and meetings (2008, 2014, and 2016) of the ECS.

Abstract:

The inositol 1, 4, 5-trisphosphate (IP3) receptors (IP3Rs) are ubiquitously expressed Ca2+ release channels of the endoplasmic

reticulum (ER). Th e resulting Ca2+ signals are instrumental in the regulation of many intracellular processes. It is now

becoming increasingly clear that contact sites between ER and mitochondria or between ER and lysosomes is very important

for controlling mitochondrial energetics, autophagy and apoptosis. Anti-apoptotic Bcl-2 leads to increased cell survival by both

blocking pro-apoptotic family members like Bax and Bak and by inhibiting IP3R mediated Ca2+ transfer to the mitochondria.

As Bcl-2 is upregulated in a variety of cancers, including diff use large B-cell lymphoma (DLBCL) and chronic lymphocytic

leukemia (CLL), we developed in collaboration with Clark W Distelhorst (Case Western Reserve University, USA) BIRD-2, a

peptide tool that disrupts Bcl-2/IP3R interaction by binding to the Bcl-2 BH4 domain, increasing IP3 induced Ca2+ release and

triggering apoptosis of DLBCL cells and of primary CLL cells. Interestingly, DLBCL cell lines with high expression levels of

the high affi nity IP3R2 isoform, were the most sensitive to BIRD-2. In addition to high levels of IP3R2 expression, constitutive

IP3 signaling downstream of the tonically active B-cell receptor increases sensitivity towards BIRD-2. Under standard growth

conditions, this constitutive IP3 signaling fulfi lled a pro-survival role, since inhibition of phospholipase C using 2.5 μM U73122

caused cell death in various DLBCL cell lines and primary CLL cells, but at lower concentrations protected against BIRD-

2-induced Ca2+ release and apoptosis. Taken together, our data indicate that on the one hand, constitutive IP3 signaling is

necessary for cancer cell survival and proliferation but that on the other hand to survive this chronic signaling the cancer cells

are dependent on high Bcl-2 levels to prevent excessive Ca2+ release via IP3Rs. Disrupting Bcl-2/IP3R interaction switches

constitutive IP3 signaling to a pro-death signal and may therefore lead to a novel therapeutic approach

Session Introduction

Ray Unwalla

Pfizer Inc, USA

Title: Structure based approach to identify potent tissue selective androgen receptor modulators

Biography:

Ray Unwalla is a computational chemist at Pfi zer, USA working in the Infl ammation and Immunology Department. He has several years of drug discovery experience

and has expertise in the area of nuclear hormone receptors, kinases and GPCRs. He has an in depth knowledge of various computational tools and techniques to

provide insightful design hypothesis and specifi c compound proposals that test design hypothesis. He was involved in project teams that successfully delivered two drug

candidates i.e., Dutasteride for benign prostate hyperplasia, Duavee for hormone replacement therapy and six clinical candidates for the progesterone, ERα LXRβ, SARM,

JAK1 and JAK3 targets.

Abstract:

The steroids testosterone and dihydrotestosterone (DHT) are androgens that play an important role in the development and

maintenance of a variety of physiological responses such as male sexual function, bone density, muscle mass and strength.

Th e androgen receptor is a nuclear hormone receptor that is expressed in many tissues and is responsible for mediating the

actions of testosterone and DHT. Patients that have defects in the androgen receptor or have androgen defi ciencies can be

eff ectively treated with exogenous testosterone and other steroidal androgens as a hormone replacement therapy. Th e anabolic

eff ects of testosterone have shown benefi t in age related decline of bone density and muscle mass. However, the side eff ect profi le

of testosterone and other currently available anabolic steroids precludes their wide spread use and the chronic administration

of steroidal androgens is associated with potential serious side eff ects such as hepatotoxicity, prostate hypertrophy and cancer.

In addition, the oral bioavailability of testosterone is poor and the route of its administration is generally through topical

formulations. We will describe our eff orts to fi nd novel series of oral tissue selective androgen receptor modulators (SARM)

i.e., cyanopyrroles and indolines that selectively promote muscle growth while showing reduced androgenic eff ects on the

prostate and seminal vesicles. Using a docking approach, we were able to delineate the binding mode of these series and further

optimize the potency. An x-ray structure of a lead compound 7 bound to AR ligand binding domain revealed an interesting

electrostatically unfavorable interaction of the 3-fl uorophenol group with a carbonyl backbone of Leu704 residue. QM based

intermolecular potential calculation performed to understand the strength of this interaction along with ligand strain energy

calculations indicate a small energy penalty of ~0.6 kcal/mol paid by the ligand to adopt the bound state conformation

R Holland Cheng

UC Davis, USA

Title: Cryo-EM and multimodal imaging towards nanoplatform design for next generation of theranostics

Biography:

R Holland Cheng is a Professor of Molecular and Cellular Biology at University of California. He received a Master degree in 1989 and a PhD degree in 1992 from Purdue

University. He has served as a Panel Reviewer for NIH programmed project grant, NIH Microbiology & Infectious Diseases Research Committee, NIH National Center

for Research Resources, Wellcome Trust, UK and Earth & Life Sciences Council, Netherlands (2002). He has actively served on the advisory board of the International

Microscopy Congress plus regional microscopy societies, American Biographical Institute, and International Virus Assembly Symposium. He is also a regular member of

the American Chemical Society, American Society for Microbiology and American Biophysical Society. He has published nearly fi fty papers in peer reviewed journals and

his research mainly focuses on proteome imaging of macromolecular systems.

Abstract:

Cryogenic electron microscopy (cryo-EM) provides a visible and quantitative approach to investigate the function of

structural proteins of viruses, both in assembly and disassembly. Th e fundamental biochemical properties of metastable

intermediates and stable mature form of viral capsids, responsible for their capacity to resist extreme pH and digestive enzyme

degradation, are revealed through cryo-EM. In this presentation, the structural functionality of Hepatitis E viral nanoparticles

(HEVNP), as a multimodal delivery platform, is assessed for the delivery of medicals or agents to target tumors through

both circulation and mucosal routes. Aided by multimodal imaging and integrative machine learning networks, the targeting,

tissue distribution, and payload packaging are investigated and demonstrated. Together, cryo-EM and cellular tomography are

exemplifi ed in the recent outcomes of unveiling the molecular mechanisms essential for platform vector design towards the

success of constructing a non-invasive, mucosal targeting system.

Wolfgang B Fischer

National Yang-Ming University, Taiwan

Title: Small viral channel forming proteins-structure based computational modeling and analysis Small viral channel forming proteins-structure based computational modeling and analysis Wolfgang B Fischer, National Yang-Ming University, Taiwan

Biography:

Abstract:

A large number of viruses encode small proteins which are expressed in the infected cell to form ion and small molecules

conducting channels. Th ese proteins are also called viroporins. For some of the viruses these proteins are essential for

infectivity cycle and thus, they comprise interesting drug targets. From the structural and mechanistic point of view, they

comprise interesting tools to study membrane protein assembly and dynamics in particular when the outcome of the assembly

is an ion conducting system. Computational tools play an important part in this investigation. Using a 2D docking tool

structural models of polytopic membrane proteins such as p7 of hepatitis C virus (HCV) with two transmembrane domains

(TMDs) and E5 of human papilloma virus – 16 (HPV-16) with three TMDs are generated in a putative functional form.

From molecular dynamics (MD) simulations of these proteins embedded in a hydrated lipid bilayer ion selectivity of these

proteins is investigated also by calculating potential of mean force (PMF) profi les of ions along the conducting pathway.

Th e calculations support weak selectivity of these channel proteins, strengthening the quality of the modeled structures. In

addition, full correlation analysis (FCA) provides insight into the dynamic of these proteins. Th e individual subunits of the

protein assemblies show asymmetric behavior during equilibrium dynamics, allowing for proposing several models of gating.

Liliane Mouawad

Institut Curie, France

Title: Effects of sarcolipin on Ca2+ pump SERCA1a enzymatic cycle studied by normal mode analysis

Biography:

Liliane Mouawad was always interested in understanding the mechanism of action of proteins or protein assemblies. This understanding may be based on either molecular

simulations or on experiments like NMR. But her expertise is primarily in molecular dynamics simulations and more precisely in normal mode analysis (NMA). She

has developed several methods going from the calculation of normal modes of very large systems or of images, to the calculation of the pathway between two protein

conformations or to the prediction of the compactness of a calcium-binding protein. Recently, she was also involved in docking and virtual screening themes, where she

has acquired enough expertise to develop a new consensus methodology to overcome some issues observed in these approaches.

Abstract:

The Ca2+ pump SERCA1a is a P-type ATPase, localized in the sarcoplasmic reticulum membrane of striated muscle cells.

SERCA1a is involved in the contraction/relaxation process by fast pumping the cytoplasmic Ca2+ into the reticulum.

Th roughout its catalytic cycle, SERCA1a presents two major conformations: the E1 conformation where its Ca2+ channel is open

toward the cytoplasm and the E2 conformation where this channel is open toward the lumen. Th is conformational transition

is enhanced by ATP phosphorylation which occurs aft er Ca2+ binding by SERCA1a. Sarcolipin (SLN), a transmembrane helix

of 31 residues regulates SERCA1a by diminishing its affi nity to Ca2+. Th e mechanism of this regulation is not elucidated yet

despite the knowledge of the crystal structure (see fi gure). To decipher this mechanism, we performed normal mode analysis

(NMA), in the all-atom model on two systems, in the presence and the absence of SLN in a POPC membrane and one layer of

water for the soluble parts of the protein. Th is analysis showed that both systems are prone to go toward the conformation of

2Ca2+ E1 more easily than toward that of E2. However, both transitions seem more diffi cult in the presence of SLN, because of

some specifi c interactions with SERCA1a that result in additional fl uctuations of SERCA1a-SLN. A long-distance transmission

of information (over 35 Å) within the protein was also observed, explaining the phosphorylation diffi culty in the complex.

Th ese results provide new insights into the mechanism of the SERCA1a enzymatic cycle and its regulation by SLN.

Sangwook Wu

1Pukyong National University, South Korea

Title: Network analysis of the conformational change of c-Src, a tyrosine kinase, by molecular dynamics simulation

Biography:

Sangwook Wu received his BA degree in Biochemistry from Yonsei University, Korea in 1990. After working as a Scientist at Samsung Display Devices during 1995-1999,

he obtained his PhD in Theoretical Condensed Matter Physics from Iowa State University during 1999-2005. He joined the Computational Biophysics Lab at UNC-Chapel

Hill (Dr. Lee Pedersen) as postdoctoral research associate during 2005-2014. From 2014 to the present, he has been a faculty member at Pukyong National University in

Korea. His research interests are in the areas of computational dynamics of biological macromolecules.

Abstract:

Non-receptor tyrosine kinase c-Src plays a critical role in numerous cellular signaling pathways. Activation of c-Src

involves a change from its inactive to the active state accompanied by large-scale conformational change depending

on the phosphorylation state of two major phosphorylation sites, Tyr416 and Tyr527. A detailed mechanism for the entire

conformational transition of c-Src via phosphorylation control of Tyr416 and Tyr527 is still elusive. In this study, we investigated

the inactive to active conformational change of c-Src by targeted molecular dynamics simulation. Based on the simulation, we

proposed a dynamical scenario for the activation process of c-Src. A detailed study of the conformational transition pathway

based on network analysis suggests that Lys321 plays a key role in the c-Src activation process.

Session Introduction

Yuri L. Lyubchenko

University of Nebraska Medical Center, USA

Title: Nanoscale dynamics of protein-DNA complexes as revealed with high-speed AFM

Biography:

Yuri L Lyubchenko is a Professor of Pharmaceutical Sciences at the University of Nebraska Medical Center, USA. His research focuses on understanding

fundamental mechanisms underlying health and disease, which is a key for developing new and more effective diagnostics and medications. This primary basic

research allows him not only to identify new drug targets for small molecule drugs, it also leads to development of the nanotools and methods to discover novel

approaches for diagnostic, treatment and disease prevention and to more rapidly determine their effi cacy at the molecular level.

Abstract:

High-speed atomic force microscopy (HS-AFM) is an advanced technique with numerous applications in biology,

particularly in molecular biophysics. Developed as a time-lapse AFM technique for direct imaging fully hydrated

biological molecules, HS-AFM is currently capable of visualizing the dynamics of biological molecules and their complexes

at a video-data acquisition rate. Spatial resolution at the nanometer level is another important characteristic of HS-AFM. Th is

presentation focuses on examples of primarily protein-DNA complexes to illustrate the high temporal and spatial resolution

capabilities of HS-AFM that have resulted in novel models and/or the functional mechanisms of these biological systems.

Emmanuelle Bignon

Danish Cancer Society, Denmark

Title: Towards an accurate description of the S-nitrosylated cysteine by classical molecular simulations: implications for the study of cancer related SNO-proteins

Biography:

Emmanuelle Bignon is a Postdoctoral researcher working in the Computational Biology Laboratory within the Danish Cancer Society Research Center, Copenhagen,

Denmark. Her research interests focus on biochemical phenomena implicated in diseases onset and development. She uses computational chemistry tools, i.e.,

all-atom molecular dynamics, quantum calculations, QM/MM(-MD) techniques, in order to investigate reactivity and dynamical behavior of DNA and disease

related proteins. Her Postdoctoral project, supported by the Alfred Benzon Foundation, deals with redox signaling mechanisms, especially S-nitrosylation (SNO)

of cysteines, a post-translational mutation which plays an important role in cancer cells. She also investigated the structure and reactivity of damaged DNA during

her PhD obtained in June 2017.

Abstract:

Nitric oxide plays an important role in the redox signaling pathway. Indeed, its reaction with cysteines, resulting in the

formation of S-nitrosothiols, has shown to be involved in a broad variety of biochemical and physiological processes in a

large variety of organisms. Large amounts of investigations have been led in order to understand the regulation mechanisms

driven by protein S-nitrosylation and their implications in cancer cells development. Th ousands of proteins have been proven

to undergo S-nitrosylation in vivo and the dysregulation of this process is implicated in various types of severe diseases,

including cancer onset, progression and treatment resistance. Th us, the understanding of S- nitrosylated proteins (SNOproteins)

structural behavior and reactivity is of utmost importance towards the development of new anti-cancer therapeutics.

Nowadays, there is a lack of information concerning the structural and electronic features of nitrosylated cysteine, with only few

NMR and x-ray structures reported for SNO proteins. In this framework, molecular modeling has been proven to be a useful

tool to obtain predictive structural and dynamical behavior of biomolecules. Th e S-nitrosocysteine (CysNO) non-canonical

amino acid exhibits a very complex chemistry, due to the presence of two antagonist resonance structures with very diff erent

electronic features. Th erefore, the accurate description of this moiety’s structure is highly challenging, and prediction of its

chemical properties has to be performed using high-level quantum methods. Unfortunately, such methods are strongly timeconsuming,

and their computational cost is prohibitive for the study of large system such as proteins. Force fi eld parameters

have been developed in order to perform classical molecular dynamics simulations of S-nitrosylated proteins to decipher their

dynamical features. Nevertheless, the accuracy of these parameters has not been extensively cross-validated and still need to be

tested. For this purpose, we performed extensive all-atom classical molecular dynamics simulations on proteins for which the

crystal structures harboring CysNO have been reported. Th e conformations sampled by all atom simulations with the two sets

of parameters have been confronted to the experimental structures in order to validate their effi ciency in reproducing CysNO

behavior. Th is checking is an important step in the design of accurate force fi eld parameters, which would allow computational

chemist to investigate the dynamical properties of S-nitrosylated proteins and their involvement in cancer cells mechanisms.

Manuela A A Ayee

University of Illinois at Chicago, USA

Title: Endothelial membrane stiffening under osmotic challenge

Biography:

Manuela A A Ayee is a Postdoctoral research fellow in the Department of Pulmonary and Critical Care Medicine at the University of Illinois at Chicago. Her current

research focuses on investigating the modulation of cellular biomechanics by oxidized lipids and sterols under hypercholesterolemic conditions. She obtained her

PhD in Chemical Engineering with a specialization in Computational Biomolecular Modeling and Interfacial Phenomena. Her broad research goals include combining

computational and experimental techniques to reveal the role of small molecules in pathogenesis and elucidating the molecular mechanisms underlying their effects

on membrane biomechanics and their subsequent modulation of cell biophysical properties. She will begin as a Professor of Chemical Engineering and Chemistry

at Dordt College, Iowa in 2018.

Abstract:

Cell volume regulation is a fundamental property of all mammalian cells. Numerous signaling pathways are known to be

activated by cell swelling and to contribute to cell volume homeostasis. Cellular biomechanics and membrane tension

have long been proposed to couple cell swelling to signaling pathways; however, the impact of swelling on these parameters

has yet to be fully elucidated. In this study, we utilize atomic force microscopy (AFM) under isotonic and hypotonic conditions

to measure the mechanical properties of human aortic endothelial membranes. From AFM force/displacement curves, we

obtain estimates of membrane elastic modulus, which refl ects the stiff ness of the sub-membrane cytoskeleton complex and the

force required for membrane tether formation, refl ecting membrane tension and membrane cytoskeleton attachment. We fi nd

that hypotonic swelling results in signifi cant stiff ening of the membrane region of endothelial cells, without a corresponding

change in membrane tension or membrane-cytoskeleton attachment. Furthermore, depolymerization of F-actin in the

cytoskeleton, which as expected results in a dramatic decrease in the cellular elastic modulus of both the membrane and the

deeper cytoskeleton, indicating a collapse of the cytoskeleton scaff old, does not abrogate swelling-induced stiff ening of the

membrane, instead this stiff ening is enhanced. We propose that the hypotonically induced membrane stiff ening should be

attributed to an increase in hydrostatic pressure that results from an infl ux of solutes and water into the cells. Most importantly,

our results suggest that increased hydrostatic pressure, rather than changes in membrane tension, could be responsible for

activating volume sensitive mechanisms in hypotonically swollen cells.

Shu-Fang Hsu

Institute of Biological Chemistry, Academia Sinica, Taiwan

Title: Hybrid methods reveal the structural architecture of PTPN3-p38 γ active-state complex

Biography:

Shu-Fang Hsu has her expertise in cell biology focusing on tyrosine phosphorylation-dependent cell signaling which coordinated by protein tyrosine kinases (PTKs)

and protein tyrosine phosphatases (PTPs). PTPs comprise a superfamily of enzymes that control a diverse array of signal transduction pathways, therefore exert

their biological functions including tumorigenicity. Her current interest is crystal structure study on PTPN3-p38γ complex for structure-based drug design as an

anticancer therapy for colon cancer.

Abstract:

PTPN3 (also known as PTPH1) cooperate to promote Ras-induced oncogenesis in human colorectal cancer. Comprehensive

structural information on the PTPN3-p38γ interaction is critical for structure-based drug design as a new means in anticancer

therapy.

Methodology & Th eoretical Orientation: In order to obtain the architecture features of PTPN3-p38γ active-state complex,

a hybrid method combining small-angle x-ray scattering (SAXS), chemical cross-linking coupled to mass spectrometry (CXMS),

hydrogen deuterium exchange mass spectrometry (HDX-MS), and x-ray crystallography were adopted.

Findings: To build the molecular architecture of PTPN3-p38γ active-state complex, the phosphatase domain of PTPN3 in its

substrate trapping mutant form was used to interact with the phosphorylated p38γ in vitro. Isothermal titration calorimetry

(iTC) analysis showed that PTPN3 binds to phosphorylated p38γ in a submicromolar affi nity, suggesting the formation

of a stable complex. CX-MS analysis unraveled the close proximity between the catalytic site of PTPN3 and the activation

loop of phospho-p38γ. HDX-MS results further demonstrated that the glutamic acid-containing loop (E-loop) and the

phosphotyrosine recognition loop (pY loop) of PTPN3 play a critical role in recruiting p38γ as a substrate during catalysis.

Preliminary crystals of PTPN3-p38γ active-state complex were obtained and the microseeding technique was applied to

optimize the crystal formation.

Conclusion & Signifi cance: Atomic structure of PTPN3-p38γ active-state complex may reveal the molecular features for the

design of new drug against the progression of colorectal cancer.

Chair

Igor Sokolov

, Tufts University, USA

Session Introduction

Igor Sokolov

Tufts University, USA

Title: Quantitative and repeatable measurements of elastic moduli of cells

Biography:

Igor Sokolov is an expert in atomic force microscopy in studying cells and biological tissues. Being initially trained as a Physicist, he received Postdoctoral training

in Microbiology. He is the recipient of the E L Ginzton International Fellowship Award from Stanford University for his work on atomic force microscopy, he received

Graham Research Award (Clarkson University), Simon Greenberg Foundation Scholarship for the study of aging skin, etc., in 2000 he joined Clarkson University,

where he achieved the title of Full Professor and served as Director of the Nano-engineering and Biotechnology Laboratories Center. He has 150+ refereed

publications, including such journals as Nature, Nature Nanotechnology, Advanced Materials, etc., He holds 20+ patents. His current research focuses on nanomechanics

of soft material, molecules and cells; atomic force microscopy; nanophotonics and the studies towards understanding of nature of cancer, early detection

of cancer based on altered biophysical properties; self-assembly

Abstract:

Mechanics of eukaryotic cells at the nanoscale is a challenging problem due to complexity of cells. Besides fundamental

interest, such measurements are important because it has been demonstrated that the elastic modulus of cells can

correlate with various diseases and even aging. Atomic force microscopy (AFM) is the technique of use. At the same time, there

is a lot of confusion in the process of measurements of the elastic modulus of cells. In the talk, author will present the modern

overview of the topic, describe step-by-step how to do the measurements of the elastic modulus in quantitative manner that is

independent of the particular microscope, AFM probe, and mechanical model.

John B Bruning

The University of Adelaide, Australia

Title: The human sliding clamp as a therapeutic target

Biography:

John B Bruning received BSc from Texas A&M University in 1997. He began crystallography in the Laboratory of Yousif Shamoo at Rice University. During his

graduate studies he worked on the structural mechanism of the human sliding clamp and its interactions with DNA replication proteins. He received his PhD in 2005

and completed two successful Post-docs; the fi rst was at the Scripps Research Institute from 2005-2007 working on structural studies of nuclear receptors including

PPAR, RXR, ER and TR and his second Post-doc was with Jim Sacchettini in the Houston Medical Centre and as a part of the TB structural genomics consortium.

He received his fi rst faculty position at the University of Adelaide as a Lecturer in 2012. He was tenured in 2015 and promoted as Senior Lecturer in 2016. Due to

his continued collaboration with Scripps Research Institute, he was also appointed as Adjunct Professor of the Scripps Research Institute in 2016.

Abstract:

The human sliding clamp (also known as PCNA) controls access to DNA of many of the proteins involved in essential

processes such as DNA replication, DNA repair and cell cycle control. Proteins compete for interaction with the PCNA

surface by means of a short, conserved peptide sequence known as the PCNA-interacting protein motif (or PIP-box). Binding

to PCNA via the PIP box allows access to DNA. For example, the major replicative polymerase, pol delta, requires PCNA for

processive DNA synthesis, without interaction with PCNA the polymerase dissociates from DNA and is incapable of processive

DNA synthesis. As such, many groups have proposed the usefulness of PIP box mimetics for use as cancer therapeutics given

they would block upregulated PCNA form allowing interaction with pol delta and hence would inhibit DNA replication.

However, no peptide mimetics of PCNA have been forthcoming to date. Here we describe the design and synthesis of the fi rst

PCNA peptidomimetic. Our mimetic, ACR2, was designed through synthetic lactam chemistry to constrain the secondary

structure of the peptide for optimized binding to PCNA. NMR solution studies show that the wild type p21 peptide from which

ACR2 was designed adopts no defi ned secondary structure in solution, while our mimetic adopts a 310 helix in solution, which

has been shown in previous studies to be essential for PIP box binding to PCNA. Binding experiments determined a KD of 200

nM of ACR2 for PCNA, which is higher than the wild type peptide. A co-crystal structure of ACR2 bound to hPCNA revealed

the mechanism of interaction of this mimetic with PCNA.

Miki Senda

High Energy Accelerator Research Organization (KEK), Japan

Title: Crystallization strategy when no crystals are obtained in the initial screening

Biography:

Miki Senda has completed her PhD from Nagaoka University of Technology in 2008. She is an Assistant Professor of Structural Biology Research Center in High

Energy Accelerator Research Organization (KEK). She has several collaborations in which she has worked as an expert of protein crystallization and crystal quality

improvement. She received Oxford Cryosystems Low Temperature Prize at the 63rd Annual meeting of the American Crystallographic Association (ACA) in 2013.

Abstract:

Protein crystallography is an indispensable tool in the pharmaceutical and biochemical sciences. Refi nements of the protein

crystallography beam lines and their integrated programs for crystal structure analysis allow us to perform automatic or

semi-automatic structural determinations using well-diff racted crystals. However, the production of well-diff racted crystals

is still a bottleneck, even when using crystallization robots and common screening kits. Th e process of protein crystallization

does not follow a standardized, routine protocol, except in the case that good crystals are obtained at an initial crystallization

screening. In the more frequent case that no crystals or only poor crystals appear at the initial screening, there is no general

consensus regarding the next step. Nonetheless, even in the absence of well-diff racted crystals at an initial screening, it is still

possible to optimize the crystallization conditions based on the accumulated data from a wide range of protein-crystallization

attempts. Th e more such crystallization data are available, the more appropriate and effi cient optimization will be possible,

since the crystallization conditions diff er for each protein. Th erefore, at our laboratories, we are currently trying to accumulate

many experiences of protein crystallization and crystal quality improvement of poor crystals through collaboration with not

only academia but also with pharmaceutical companies. Here, we present our strategy for the effi cient generation of good

quality crystals and the successful application of our crystal quality improvement method to histone chaperone TAF-Ibeta,

the CagA oncoprotein from Helicobacter pylori and GTP sensor PI5P4Kbeta. We believe that our strategy will be applicable to

other proteins as well.

Thomas Prevenslik

QED Radiations Discovery Bay, China

Title: Protein folding and unfolding by quantum mechanics

Biography:

Thomas Prevenslik is a retired American living in Hong Kong and Berlin. Because classical physics does not work at the nanoscale, he has developed the theory of

QED radiation based on quantum mechanics. He developed the simple theory of QED based on the Planck law of QM. Differing from the complex QED by Feynman

and others, simple QED assumes any heat absorbed at the nanoscale having high surface-to-volume ratios place interior atoms under high EM confi nement that

by the Planck law of QM precludes the atoms from having the heat capacity to conserve heat by an increase in temperature. In the instant topic of protein folding

and unfolding by quantum mechanics, the atoms may only conserve heat by creating EM radiation that by removing electrons by the photoelectric effect charges

the atoms positive inducing Coulomb repulsion that enhances unfolding. On a picosecond time scale, the electrons recombine with charged atoms to place the

protein under van der Waals attraction that fold the protein. Driven by heat, protein folding, and unfolding is the consequence of fl uctuations between QM induced

charge and neutral states.

Abstract:

Proteins are sensitive to electrostatic charges from amino acid side chains that change with conformation. But molecular

dynamics (MD) simulations of protein folding and unfolding are based on classical force fi elds with electrostatic interaction

represented by fi xed point charges. Quantum mechanics (QM) modifi cation of point charges during conformational changes is

required but is impractical because of computational costs. Computation costs aside, even if point charges were continuously

updated, the eff ect on protein folding and unfolding would be insignifi cant compared to the more fundamental QM eff ect

of the Planck law on the heat capacity of atoms. In this regard, proteins are generally thought to unfold upon increasing

temperature based on the classical assumption the constituent atoms have heat capacity. But the Planck law requires the heat

capacity of the atom to vanish with conservation proceeding by creating EM radiation that by the photoelectric eff ect removes

electrons to positively charge the protein atoms. What this means is the heat thought to induce unfolding by increasing the

temperature of proteins is actually conserved by producing charge that unfolds the protein by Coulomb repulsion. To illustrate

QM induced charge, the MD simulation of folding and unfolding using for a simple 5-atom protein is illustrated in Figure 1.

Initially, the protein in the form of a semi-circle relaxes under L-J forces but does not unfold. L-J stands for Lennard-Jones.

Unfolding occurs upon applying QM induced repulsive positive charge (0.5-1 electron charges) on each atom. Folding back to

an intermediate cluster occurs by relaxing the protein with L-J forces alone without QM induced charge. Th e protein returns

to an inverted semi-circular shape by unfolding the cluster by applying the QM induced repulsive charge. How the protein

constantly modifi es QM induced charge is discussed.

Chair

Peter B Stathopulos,

University of Western Ontario, Canada

Session Introduction

Fabrice Gorrec

Fabrice Gorrec

Title: The Morpheus III protein crystallization screen: at the frontier of drug discovery

Biography:

Abstract:

The original Morpheus screen has proven to be a very effi cient protein crystallization screen in the long term for a broad

variety of protein samples and the fi rst follow-up screen, Morpheus II starts to have an impact for many research groups.

Th e main ideas behind the formulation of Morpheus III were the same as originally to increase the chances of crystal nucleation

and growth by integrating mixes of additives that can act as stabilizers, cross-linkers, etc. Follow systematic approaches to select

the reagents and formulate the screen, such as the integration of ligands that are highly represented in the PDB and a 3D grid

formulation. Consider pragmatic ways to facilitate structure solution. For example, the screening of cryoprotectants and fl ashfreezing

crystals are simplifi ed. Th e novelty in Morpheus III is the selection of small drug-like compounds as additives (average

MW=248 Da). To some extent, the approach can be compared to fragment-based lead discovery. Th e primarily aim however is

to obtain novel macromolecular crystals (with or without ligand observed in the structures). Th e fi nal formulation of the new

96-condition crystallization screen integrates 44 compounds overall, divided into 8 mixes of additives. Each mix of additives is

combined with 4 cryo-protected precipitant mixes and 3 buff er systems to form the 3D grid.

Vesa P Hytönen

1University of Tampere, Finland

Title: Steered molecular dynamics and single molecule atomic force microscopy to explore the mechanical response of talin

Biography:

Vesa P Hytönen is a Head of the Protein Dynamics research group in Faculty of Medicine and Life Sciences at the University of Tampere, Tampere, Finland. After

graduating PhD from the University of Jyväskylä, Jyväskylä, Finland at 2005, he conducted Postdoctoral training at ETH Zurich, Switzerland during 2005–2007. He then

continued as a Postdoctoral researcher at the University of Tampere and established independent research group at 2010. He is currently working as Associate Professor

at the University of Tampere. His research interests are mechanobiology, protein engineering and vaccine research and he has authored more than 100 scientifi c articles.

Abstract:

Talin is cytoplasmic protein connecting integrin receptors to actomyosin network. Th is linkage is essential for cell anchoring

and spreading and naturally also for development. Talin acts as a hub for molecular interactions in focal adhesions and

the interactions between talin and binding partners are regulated by mechanical signals. We study the mechanical response of

talin by steered molecular dynamics. Both constant force and constant velocity simulations in explicit water have been found

useful to explore the molecular features of talin. We have found talin rod subdomains to diff er from each other in terms of their

mechanical stability. Importantly, we have been able to compare and validate the results by using experimental data obtained

with single-molecule atomic force microscopy. Destabilizing point mutations applied on talin rod have been found to cause

signifi cant changes in cell spreading, migration and cellular traction force. Our recent studies focus on mechanically weakened

talin forms, intermediates of protein unfolding and engineering of unfolding-resistant talin forms.

Fabrice Gorrec,

MRC Laboratory of Molecular Biology, UK

Title: Automated protocols for macromolecular crystallization at the MRCLaboratory of Molecular Biology

Biography:

Fabrice Gorrec has participated in the development of innovative technologies applied to protein crystallization over 14 years, including microplates (e.g., TOPAZ®),

liquid-handlers (e.g., Dragonfl y®) and initial screens (e.g., MORPHEUS™). Since 2008, he is responsible for the crystallization facility at the MRC Laboratory of Molecular

Biology (MRC-LMB, Cambridge, UK) where he invented and developed the 96 condition Morpheus protein crystallization screens. He pursued his Master’s degree in

Molecular Biology, Biochemistry and Biophysics from University of Rennes, France.

Abstract:

When high quality crystals are obtained that diff ract x-rays, the crystal structure may be solved at near atomic resolution.

Th e conditions to crystallize proteins, DNAs, RNAs and their complexes can however not be predicted. Employing

a broad variety of conditions is a way to increase the yield of quality diff raction crystals. Two fully automated systems have

been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization

screening against 1,920 initial conditions by vapor diff usion in nano liter droplets. Semi-automated protocols have also

been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that

potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefl y

discussed. Taken together, they enable convenient and highly effi cient macromolecular crystallization in a multi-user facility,

while giving the users control over key parameters of their experiments.

Raphael Taiwo Aruleba

University of Zululand, South Africa

Title: Development of new and effective anti-schistosomal drugs using antimicrobial peptides as lead compounds: A computer-aided drug design study

Biography:

Raphael Taiwo Aruleba is an Advocator for human and renders selfl ess service especially in the fi eld of science to humanity. Growing up he envisioned making positive

impact on many people as possible, which ultimately led him to the discovery of anti-microbial peptides that can be used in tackling Schistosomiasis using various

bioinformatic techniques. He also used ULBP2 to study cancer cells and Ganoderma lucidum to inhibit the growth of Plasmodium berghei in mice.

Abstract:

With the exponential increase in the prevalence of schistosomiasis, Praziquantel (PZQ) remains the only eff ective drug

in the anti-schistosomal arsenal. However, no signifi cant approaches have been made in recent years in the discovery

of new anti-schistosomal drugs, even with widely-reported resistance of the schistosome worm to PZQ over very large foci.

Th erefore, it is imperative to develop a new drug against this debilitating disease using the broad-spectrum therapeutic potentials

of antimicrobial peptides (AMPs). AMPs are natural antibiotics produced by all living species; they have multifunctional

properties and are currently explored as a vital source for the development of new drugs. In this study, six putative AMPs

(TAK1-TAK6) were identifi ed to possess anti-schistosomal capabilities. Added to this, glycosyltransferase and axonemal

dynein intermediate chain schistosomal proteins were identifi ed using in silico methods as vital proteins for the survival of the

parasite in the host. Th e 3D structures of the AMPs and the proteins were modelled using the I-TASSER, while Patch Dock

was employed to ascertain the interaction between these schistosome proteins and the AMPs. Results obtained show the

putative AMPs have good binding affi nity to the schistosomal proteins. So, TAK3 and TAK6 showed highest binding affi nities

to glycosyltransferase and axonemal dynein intermediate chain respectively. On the whole, all generated AMPs are potential

therapeutics target that could be further developed as drug candidates in the fi ght against schistosomiasis and could as well

prove eff ective against PZQ resistant schistosome strains.

Patrick Carl

Bruker Biospin, Germany

Title: Probing structures, interactions and rearrangements by EPR

Biography:

Electron Paramagnetic Resonance (EPR) is a powerful technique used to explore biological macromolecular structures.

Using intrinsic or extrinsic EPR species, the specifi cs of structural topology, protein-protein/protein-RNA interactions and

structural rearrangements are observed. Continuous wave EPR is used to probe the local environment to obtain information

such as pH, viscosity, rotational correlation time, and hydrophobicity. Th is information can easily be obtained using a user

friendly benchtop spectrometer, the EMXnano. Pulse EPR opens the possibility to probe more specifi cally the interactions in

the biological structures. Double Electron Electron Resonance (DEER) permits the direct distance determination from 2 nm

up to 10 nm. Th e DEER technique is not size limited and aides in the determination of multidomain protein and nucleic acid

structures.

Abstract: