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          Labs @ NIAMS
          Protein Expression Laboratory








Protein Expression Laboratory


Paul Wingfield, Ph.D. FacebookEmailPrintFacebookEmail
        Protein Expression Lab

      Overview

                                  Senior Investigator
      
  


Paul Wingfield, Ph.D.
      
  


            wingfiep@mail.nih.gov
      
                                                                        

  
   Research Areas (IRP Lab Groups)
          Immunology,
          Microbiology and Infectious Diseases,
          Molecular Biology and Biochemistry,
          Virology
                                
            The NIAMS Protein Expression Laboratory (PEL) studies the structure and function of viral proteins, primarily those of the Human Immunodeficiency Virus (HIV-1) and the Hepatitis B Virus (HBV). The PEL also studies the interactions of these viral proteins with host proteins.

Most structural biology techniques, especially those for studying the three-dimensional structures of proteins, require large quantities of highly purified, monodisperse, and correctly folded proteins. The PEL uses recombinant DNA techniques to produce proteins both for in-house use and for collaborating scientists. It employs techniques such as analytical ultracentrifugation, UV-CD, NMR, X-ray crystallography, and cryo-electron microscopy to characterize and determine the structures of these proteins and their complexes.

**HIV-1:**

**gp41** **and gp120:** HIV-1 entry into cells is initiated when a trimeric complex on the virion surface, composed of gp41 and gp120, interacts with the principal cellular receptor, CD4. The trimeric complex is therefore a target for vaccine and drug development. When gp120 binds to the CD4 receptor it changes the conformation of gp41, which induces fusion between the viral and host membranes. Using both NMR and X-ray crystallography, the PEL and its collaborators have determined the structure of gp41. Based on this structural and biochemical information, a mechanism for membrane fusion has been proposed.

**Protease:**Early in HIV-1 infection, viral RNA is translated into a polyprotein which is then cleaved by the viral protease at nine sites to create the functional proteins. In this process, the dimeric protease catalyzes its own production. Without the protease, viral replication is not possible and so it is an important drug target. The virus evolves to evade these drugs, lessening their efficacy. Based on the study of protease residues susceptible to the oxidative process, namely the sulfur-containing residues methionine and cysteine, the PEL and its collaborators have proposed that drugs that target the region between the subunits may provide an alternate site of intervention.
Fig. 2. Tubulin - Cryptophycin-1 ring-complexes, colored according to hydrophobicity.Fig. 1. A model of the Rev-RRE complex, colored to distinguish individual components.**Rev:** The Rev protein is an essential viral regulator that assembles on a highly structured 350-nucleotide motif, the Rev Response Element (RRE), in un-spliced and partially spliced viral transcripts, enabling their nuclear export. Despite over 30 years of active research, the molecular details whereby Rev binds to and assembles on the RRE remain largely unknown. Structural details of this complex nucleoprotein assembly would open up new drug targets. A model of the RRE, with a Rev tetramer docked onto it in accordance with X-ray crystallography, cryo-EM, NMR, SAXS, SHAPE, and mutational analyses, has been developed by the PEL ( *see Fig. 1*). The structure of an RNA aptamer that can inhibit HIV-1 by blocking Rev-RRE binding and Rev-Rev association has been determined. Engineered antibodies, their fragments (Fabs), and cyclic peptides based on the antibody CDR loops, that inhibit HIV-1 have been devised. A patent application has been filed. Rev also interacts with several host proteins which may present therapeutic targets. These include the nuclear regulatory factors B23 and Nap1, and tubulin, which are all being studied by the PEL.
Fig. 2. Tubulin - Cryptophycin-1 ring-complexes, colored according to hydrophobicity.Fig. 2. Tubulin - Cryptophycin-1 ring-complexes, colored according to hydrophobicity.**Rev and Tubulin:**The PEL has found that Rev interacts strongly and stoichiometrically with tubulin to form double-ring complexes *in vitro*. The PEL has also shown that treatment of tubulin with Cryptophycin-1 (a compound derived from marine cyanobacteria of the genus *Nostoc* with anticancer activity 1000-fold greater than paclitaxel) forms highly stable rings composed of eight tubulin dimers and that Rev binds to these to also form double-rings. The binding sites on tubulin, and the mechanisms of ring formation, are not understood for either Cryptophycin-1 or Rev. The PEL is studying the structure of these complexes by cryo-electron microscopy using the Krios microscope at the NIH Multi Institute Cryo-EM Facility (MICEF) ( *see Fig. 2*****). The PEL has now also formed complexes of Hela cell tubulin with Cryptophycin-52, an analog of Cryptophycin-1 that has previously advanced to phase II clinical trials as a chemotherapeutic. This will allow us to understand the mechanisms of these anticancer drugs and how they can be improved, something of longstanding interest to both the tubulin and cancer research communities.

**Hepatitis B Virus:**

**HBV core-antigen:** HBV infects approximately one-third of the human population, is the major cause of liver cancer worldwide and causes almost 1 million deaths annually. Although a vaccine has been developed, it is not universally available and, as the virus is vertically transmitted from mothers to infants, HBV is often acquired in childhood and becomes a chronic condition.
Fig. 3. Structure of the HBV T=3 and T=4 capsids, colored according to symmetry.Fig. 3. Structure of the HBV T=3 and T=4 capsids, colored according to symmetry.The viral capsid plays an important structural and metabolic role in the replication cycle of any virus and, being distinct from host components, is a potential target for intervention. In the case of HBV, the dimeric capsid protein assembles into shells of two sizes, called T=3 and T=4 in reference to their symmetry ( see Fig. 3 ). The PEL studies the assembly and structure of these capsids using recombinant proteins, and by making extensive use of mutagenesis and structural analysis by X-ray crystallography and electron microscopy. The PEL also studies the binding to capsids of HBV-patient and monoclonal antibodies and it has engineered antibody fragments (Fab and scFv) that effectively block capsid assembly.

**HBV e-antigen:** The viral e-antigen is a dimeric protein that subverts the human immune system to establish a chronic infection. The mechanism is not known. The e-antigen polypeptide is essentially identical to that of the core-antigen except that it has a 10-residue propeptide. The PEL has found that this difference causes the dimer to adopt a radically different arrangement from that of core-antigen and that it can be converted to an arrangement like the latter by reduction.

The PEL has produced a panel of chimeric (human framework) anti-e-antigen rabbit antibodies (Fabs), some with very high affinities (*K*d = 10-12 M). These antibodies have both diagnostic and therapeutic potential. The PEL has developed a quantitative assay for the e-antigen which is superior to existing commercial assays both in terms of sensitivity and specificity. Using these antibodies, the PEL has immunoaffinity-purified e-antigen from individual patient plasmas and subjected it to proteomic analysis, demonstrating previously unknown heterogeneity in this antigen. The X-ray crystallographic structures of several of the chimeric antibodies (as scFv) complexed with e-antigen have been determined. These antibodies bind to epitopes forming parts of the dimer-dimer interface which mediates capsid assembly in the core-antigen (and reduced e-antigen). This and future work will provide a structural framework to test the strategy of treating chronic HBV using antibodies (and derived mimics) that target both the e-antigen and capsid assembly. A patent application has been filed. The nature of these antibodies (recombinant source, human framework, exceptionally high affinity, and specificity) also raises the potential for their use as antibody-drug complexes (ADCs) in the treatment of HBV infection.
      

                                            Core Research Facilities

Labs at the NIAMS are supported by the following state-of-the-art facilities and services:

Biodata Mining and Discovery
	Flow Cytometry
	Genomic Technology
	Laboratory Animal Care and Use
	Light Imaging
	Translational Immunology

                                    Staff
                            
            **Paul Wingfield, Ph.D.**Senior Investigatorwingfiep@mail.nih.gov
    **Elif Eren, Ph.D.**Staff Scientistelif.eren@nih.gov
    **Felipe Montecinos, Ph.D.**Contractor
    **Ira W. Palmer**Research Assistantpalmeri@mail.nih.gov
    **Norman R. Watts, Ph.D.**Staff Scientistnorman_watts@nih.gov
            
                                                Image & Media Gallery
              
            Hela tubulin induced by the binding of Cryptophycin 52 (hydrophobicity surface)
Hela tubulin induced by the binding of Cryptophycin 52 (hydrophobicity surface)
        ×
      Ring of Hela tubulin
        Ring of Hela tubulin induced by the binding of Cryptophycin 52, a potent anti-cancer drug. The tubulin is rendered as a hydrophobicity surface (blue, hydrophilic; orange, hydrophobic) with Cryptophycin 52 colored magenta. The chemical structure of Cryptophycin 52 is shown in the center.
    Hela tubulin induced by the binding of Cryptophycin 52 (electrostatic surface)
Hela tubulin induced by the binding of Cryptophycin 52 (electrostatic surface)
        ×
      Ring of Hela tubulin
        Ring of Hela tubulin induced by the binding of Cryptophycin 52, a potent anti-cancer drug. The tubulin is rendered as an electrostatic surface (blue, electropositive; red, electronegative) with Cryptophycin 52 colored green. The chemical structure of Cryptophycin 52 is shown in the center.
    HIV Rev dimer helix
HIV Rev dimer helix
        ×
      computer visualization of a molecular structure
        HIV-1 Rev dimers in a helical arrangement showing their multiple interactions.
    HIV Rev polymer shown from end and side.
HIV Rev polymer shown from end and side.
        ×
      Computer visualization of the side and end of a molecular tube structure
    HIV Rev dimers associated via their ‘A’ interfaces.
HIV Rev dimers associated via their ‘A’ interfaces.
        ×
      Computer visualization of molecular structure, colored to highlight interactions
        HIV-1 Rev dimers showing their interactions via their oligomerization domains.
    A model of the Rev-RRE complex
A model of the Rev-RRE complex
        ×
      A model of the Rev-RRE complex, colored to distinguish individual components.
        A model of the Rev-RRE complex, colored to distinguish individual components.
    Tubulin-Cp1 ring
Tubulin-Cp1 ring
        ×
      Tubulin - Cryptophycin-1 ring-complexes, colored according to hydrophobicity.
        Tubulin - Cryptophycin-1 ring-complexes, colored according to hydrophobicity.
    Structure of the HBV T=3 and T=4 capsids
Structure of the HBV T=3 and T=4 capsids
        ×
      Structure of the HBV T=3 and T=4 capsids, colored according to symmetry.
        Structure of the HBV T=3 and T=4 capsids, colored according to symmetry.
    Separating Proteins by Molecular Weight
Separating Proteins by Molecular Weight
        ×
      This photograph shows Anastassia Tikhonova B.S. using electropheresis apparatus to separate proteins by molecular weight.
        Separating Proteins by Molecular Weight

This photograph shows Anastassia Tikhonova, B.S., using electropheresis apparatus to separate proteins by molecular weight. The work in this laboratory is directed towards understanding how allergenic agents and immune complexes activate one particular family of receptors (Fc receptors) and, through them, ultimately control the genes that produce the signals (cytokines) that in turn recruit other cells in the inflammatory response.

Photographer: Rhoda Baer
            
                        
                        
                                    Scientific Publications
              Selected Recent Publications
                  
      Development of an aerosol intervention for COVID-19 disease: Tolerability of soluble ACE2 (APN01) administered via nebulizer.

    Shoemaker RH, Panettieri RA Jr, Libutti SK, Hochster HS, Watts NR, Wingfield PT, Starkl P, Pimenov L, Gawish R, Hladik A, Knapp S, Boring D, White JM, Lawrence Q, Boone J, Marshall JD, Matthews RL, Cholewa BD, Richig JW, Chen BT, McCormick DL, Gugensberger R, Höller S, Penninger JM, Wirnsberger G
        

                    PLoS One.
        
                    2022;
        
                    17(7).
        
                    doi: 10.1371/journal.pone.0271066
                            PMID: 35816490
              Structural characterization of the Myxococcus xanthus encapsulin and ferritin-like cargo system gives insight into its iron storage mechanism.

    Eren E, Wang B, Winkler DC, Watts NR, Steven AC, Wingfield PT
        

                    Structure.
        
                    2022 Apr 7;
        
                    30(4).
        
                    doi: 10.1016/j.str.2022.01.008
                            PMID: 35150605
              Conformational changes in tubulin upon binding cryptophycin-52 reveal its mechanism of action.

    Eren E, Watts NR, Sackett DL, Wingfield PT
        

                    J Biol Chem.
        
                    2021 Oct;
        
                    297(4).
        
                    doi: 10.1016/j.jbc.2021.101138
                            PMID: 34461087
              Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

    Davis DA, Bulut H, Shrestha P, Yaparla A, Jaeger HK, Hattori SI, Wingfield PT, Mieyal JJ, Mitsuya H, Yarchoan R
        

                    mBio.
        
                    2021 Aug 31;
        
                    12(4).
        
                    doi: 10.1128/mBio.02094-21
                            PMID: 34399606
              Capsids of hepatitis B virus e antigen with authentic C termini are stabilized by electrostatic interactions.

    Watts NR, Palmer IW, Eren E, Steven AC, Wingfield PT
        

                    FEBS Lett.
        
                    2020 Mar;
        
                    594(6).
        
                    doi: 10.1002/1873-3468.13706
                            PMID: 31792961
              Expression of quasi-equivalence and capsid dimorphism in the Hepadnaviridae.

    Wu W, Watts NR, Cheng N, Huang R, Steven AC, Wingfield PT
        

                    PLoS Comput Biol.
        
                    2020 Apr;
        
                    16(4).
        
                    doi: 10.1371/journal.pcbi.1007782
                            PMID: 32310951
              Protein Stability and Functional Characterization of Intra-Melanosomal Domain of Human Recombinant Tyrosinase-Related Protein 1.

    Dolinska MB, Young KL 2nd, Kassouf C, Dimitriadis EK, Wingfield PT, Sergeev YV
        

                    Int J Mol Sci.
        
                    2020 Jan 3;
        
                    21(1).
        
                    doi: 10.3390/ijms21010331
                            PMID: 31947795
              The TYRP1-mediated protection of human tyrosinase activity does not involve stable interactions of tyrosinase domains.

    Dolinska MB, Wingfield PT, Young KL 2nd, Sergeev YV
        

                    Pigment Cell Melanoma Res.
        
                    2019 Nov;
        
                    32(6).
        
                    doi: 10.1111/pcmr.12791
                            PMID: 31077632
              Structures of Hepatitis B Virus Core- and e-Antigen Immune Complexes Suggest Multi-point Inhibition.

    Eren E, Watts NR, Dearborn AD, Palmer IW, Kaufman JD, Steven AC, Wingfield PT
        

                    Structure.
        
                    2018 Oct 2;
        
                    26(10).
        
                    doi: 10.1016/j.str.2018.06.012
                            PMID: 30100358
              Structure of an RNA Aptamer that Can Inhibit HIV-1 by Blocking Rev-Cognate RNA (RRE) Binding and Rev-Rev Association.

    Dearborn AD, Eren E, Watts NR, Palmer IW, Kaufman JD, Steven AC, Wingfield PT
        

                    Structure.
        
                    2018 Sep 4;
        
                    26(9).
        
                    doi: 10.1016/j.str.2018.06.001
                            PMID: 30017564
              A new HIV-1 Rev structure optimizes interaction with target RNA (RRE) for nuclear export.

    Watts NR, Eren E, Zhuang X, Wang YX, Steven AC, Wingfield PT
        

                    J Struct Biol.
        
                    2018 Aug;
        
                    203(2).
        
                    doi: 10.1016/j.jsb.2018.03.011
                            PMID: 29605570
              HIV-1 gp41 transmembrane oligomerization monitored by FRET and FCS.

    Schroeder S, Kaufman JD, Grunwald M, Walla PJ, Lakomek NA, Wingfield PT
        

                    FEBS Lett.
        
                    2018 Mar;
        
                    592(6).
        
                    doi: 10.1002/1873-3468.13010
                            PMID: 29453892
              Membrane-associated human tyrosinase is an enzymatically active monomeric glycoprotein.

    Kus NJ, Dolinska MB, Young KL 2nd, Dimitriadis EK, Wingfield PT, Sergeev YV
        

                    PLoS One.
        
                    2018;
        
                    13(6).
        
                    doi: 10.1371/journal.pone.0198247
                            PMID: 29870551
              IL-12p35 induces expansion of IL-10 and IL-35-expressing regulatory B cells and ameliorates autoimmune disease.

    Dambuza IM, He C, Choi JK, Yu CR, Wang R, Mattapallil MJ, Wingfield PT, Caspi RR, Egwuagu CE
        

                    Nat Commun.
        
                    2017 Sep 28;
        
                    8(1).
        
                    doi: 10.1038/s41467-017-00838-4
                            PMID: 28959012 
                            See all publications on PubMed
            
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        Last Updated: August 2022
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