Vakonakis Lab

Structural biology of large cellular assemblies

 

Centriole assembly and ultrastructure

 

Centrioles

Centrioles, the major component of animal centrosomes, are conserved cell organelles that duplicate once per cell cycle in order to maintain their number. Abnormalities in the centriole duplication process can lead to disease, including primary microcephaly and cancer in humans. At the same time, centrioles are very large (~400x200nm in humans) protein assemblies that seem to have few distinct structural components (SAS-6, STIL, CPAP, Cep135 and microtubules in humans). It is thus both important and intriguing to understand how centrioles form, and what are the mechanistic roles of their components. In this effort we are joined by the groups of Profs. Pierre Gönczy (cell biology, EPFL, Switzerland) and  Michel Steinmetz (biophysics of microtubules, Paul Scherrer Institute, Switzerland).

SAS-6 forms oligomers at the core of centrioles that determine the overall 9-fold symmetry of this organelle. Using a combination of X-ray crystallography and electron microscopy we have distinguish two different oligomeric forms of SAS-6. The first,  a 9-fold symmetric ringwas resolved from Chlamydomonas reinhardtii but appears common in most organisms. The second,  a 9-fold symmetric double spiral, appears restricted to the nematode worms. However, whether as spirals or rings, SAS-6 has the same function of forming an initial framework for subsequent centriole components.

"Ring or spiral? Chlamydomonas reinhardtii SAS-6 forms rings in vitro (top: electron micrograph on left, crystallographic model on right), whereas SAS-6 from Caenorhabditis elegans forms long spiral structures (bottom: top: electron micrograph on left, crystallographic model on right)."

 

Recent studies have shed light on what other centriole proteins may look like. Crystallographic studies of CPAP showed that it adopts a unique domain fold, with periodicity that matches that of tubulin in microtubules. We postulate that fibrils of CPAP may provide a molecular ruler for elongating centrioles. 

 

SAS-5, the nematode equivalent of STIL, showed on the other hand a complex arrangement comprising two oligomerisation domains. Overall SAS-5 is a hexamer, and we believe it may form a seeding point from where SAS-6 oligomers emanate.

 

Relevant publications:

Bianchi S, Rogala KB, Dynes NJ, Hilbert M, Leidel SA, Steinmetz MO, Gönczy P, Vakonakis I (2018) Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly. Mol Biol Cell 29, 722-735.

Cutts EE, Inglis A, Stansfeld PJ, Vakonakis I, Hatzopoulos GN (2016) The centriolar protein CPAP G-box: an amyloid fibril in a single domain. Biochem Soc Trans 43, 838-43.

Rogala KB, Dynes NJ, Hatzopoulos GN, Yan J, Pong SK, Robinson CV, Deane CM, Gönczy P, Vakonakis I (2015) The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation. eLife 4, 10.7554/eLife.07410.

Hatzopoulos GN, Erat MC, Cutts E, Rogala KB, Slater LM, Stansfeld PJ, Vakonakis I (2013) Structural analysis of the G-box domain of the microcephaly protein CPAP suggests a role in centriole architecture. Structure 21, 2069-77.

Hilbert M, Erat MC, Hachet V, Guichard P, Blank ID, Flückiger I, Slater L, Lowe ED, Hatzopoulos GN, Steinmetz MO, Gönczy P, Vakonakis I (2013) The Caenorhabditis elegans centriolar protein SAS-6 can form a spiral that is consistent with imparting a 9-fold symmetry. Proc. Natl. Acad. Sci. U.S.A. 110, 11373-8.

Kitagawa D, Vakonakis I, Olieric N, Hilbert M, Keller D, Olieric V, Bortfeld M, Erat MC, Flückiger I, Gönczy P, Steinmetz MO (2011) Structural Basis of the 9-Fold Symmetry of Centrioles. Cell. 144, 364-75.

 

P. falciparum exported proteins, "knobs" and malaria cytoadherence

 

 

Malaria

Malaria is a parasitic disease virtually absent in the developed world, but that still affects developing countries strongly. Most malaria deaths are caused by a single parasite species, Plasmodium falciparum, and many can be attributed to obstruction of small blood vessels in tissues by red blood cell clumps. Cytoadherence of infected erythrocytes is mediated by a system unique to P. falciparum that includes the PfEMP1 protein family and other parasite components.

Our research focuses on how parasite proteins exported to the host cell endow the erythrocyte with adhesion characteristics, as well as alter its shape and mechanical properties. Overall the parasite exports ~500 proteins, many of which are unique to Plasmodia. Despite being putative targets for therapeutic intervention, ~75% of these proteins have no functional annotation. We try to understand their function by studying the interactions between parasite-parasite and parasite-host components using structural biology and biophysics. We collaborate closely with Prof. Hans-Peter Beck (cell biology, SwissTPH) in these efforts.

We previously determined the structure of the intracellular domain of PfEMP1, the main parasite adhesion receptor, by NMR and SAXS. This segment, termed ATS, mediates adhesion forces from outside the infected erythrocyte to the cytoskeleton. Our analysis suggested that ATS is largely flexible in solution with only a single well-folded core. Yet, ATS is highly conserved. We showed that some of the conserved but flexible segments of ATS form complexes with members of the parasite PHIST protein family, and determined the first structures and complexes of PHIST proteins. A specific PHIST member, PFE1605w (also known as LyMP) was shown to strengthen cytoadherence by linking the PfEMP1 adhesion receptor with the erythrocyte cytoskeleton. 

 

We currently study how parasite components interact with the erythrocyte cytoskeleton, and in so doing form protrusions on the host cell surface (knobs) on which PfEMP1 locates. We use computational modelling, as well as more traditional schematics, to visualise what knobs look like. 

 

Relevant publications

Cutts EE, Laasch N, Reiter DM, Trenker R, Slater LM, Stansfeld PJ, Vakonakis I (2017) Structural analysis of P. falciparum KAHRP and PfEMP1 complexes with host erythrocyte spectrin suggests a model for cytoadherent knob protrusions. PLoS Pathog 13, e1006552.

 

Oberli A, Zurbrügg L, Rusch S, Brand F, Butler ME, Day JL, Cutts EE, Lavstsen T, Vakonakis I, Beck HP (2016) Plasmodium falciparum PHIST Proteins Contribute to Cytoadherence and Anchor PfEMP1 to the Host Cell Cytoskeleton. Cell Microbiol 18, 1415-28.

 

Warncke JD, Vakonakis I, Beck HP (2016) Plasmodium Helical Interspersed Subtelomeric (PHIST) Proteins, at the Center of Host Cell Remodeling. Microbiol Mol Biol Rev 80, 905-27.

 

Watermeyer JM, Hale VL, Hackett F, Clare DK, Cutts EE, Vakonakis I, Fleck RA, Blackman MJ, Saibil HR (2016) A spiral scaffold underlies cytoadherent knobs in Plasmodium falciparum-infected erythrocytes. Blood 127, 343-51.

 

Oberli A, Slater LM, Cutts E, Brand F, Mundwiler-Pachlatko E, Rusch S, Masik MFG, Erat MC, Beck HP, Vakonakis I (2014) A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface. FASEB J 28, 4420-4433.

 

Mayer C, Slater L, Erat MC, Konrat R, Vakonakis I (2012) Structural analysis of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope. J Biol Chem. 287, 7182-9.

Publications

  1. Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly.
    Bianchi S, Rogala KB, Dynes NJ, Hilbert M, Leidel SA, Steinmetz MO, Gönczy P, Vakonakis I.
    Mol Biol Cell 2018, 29, 722-735.
  2. Structural analysis of P. falciparum KAHRP and PfEMP1 complexes with host erythrocyte spectrin suggests a model for cytoadherent knob protrusions.
    Cutts EE, Laasch N, Reiter DM, Trenker R, Slater LM, Stansfeld PJ, Vakonakis I.
    PLoS Pathog. 2017, 13, e1006552.
  3. Coupling Form and Function: How the Oligomerisation Symmetry of the SAS-6 Protein Contributes to the Architecture of Centriole Organelles.
    Ford JE, Stansfeld PJ, Vakonakis I.
    Symmetry 2017, 9, 74.
  4. Plasmodium Helical Interspersed Subtelomeric (PHIST) Proteins, at the Center of Host Cell Remodeling.
    Warncke JD, Vakonakis I, Beck HP.
    Microbiol Mol Biol Rev. 2016, 80, 905-27.
  5. Plasmodium falciparum PHIST proteins contribute to cytoadherence and anchor PfEMP1 to the host cell cytoskeleton.
    Oberli A, Zurbrügg L, Rusch S, Brand F, Butler ME, Day JL, Cutts EE, Lavstsen T, Vakonakis I, Beck HP.
    Cell Microbiol 2016, 18, 1415-28.
  6. A spiral scaffold underlies cytoadherent knobs in Plasmodium falciparum-infected erythrocytes.
    Watermeyer JM, Hale VL, Hackett F, Clare DK, Cutts EE, Vakonakis I, Fleck RA, Blackman MJ, Saibil HR.
    Blood 2016, 21, 343-51.
  7. The centriolar protein CPAP G-box: an amyloid fibril in a single domain.
    Cutts EE, Inglis A, Stansfeld PJ, Vakonakis I, Hatzopoulos GN.
    Biochem Soc Trans 2015, 43, 838-43.
  8. The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation.
    Rogala KB, Dynes NJ, Hatzopoulos GN, Yan J, Pong SK, Robinson CV, Deane CM, Gönczy P, Vakonakis I.
    Elife 2015, 4, doi: 10.7554/eLife.07410.
  9. Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface.
    Oberli A, Slater LM, Cutts E, Brand F, Mundwiler-Pachlatko E, Rusch S, Masik MFG, Erat MC, Beck HP, Vakonakis I.
    FASEB J 2014, 28, 4420-33.
  10. Structural Analysis of the G-Box Domain of the Microcephaly Protein CPAP Suggests a Role in Centriole Architecture.
    Hatzopoulos GN, Erat MC, Cutts E, Rogala KB, Slater LM, Stansfeld PJ,Vakonakis I.
    Structure 2013, 21, 2069-77.
  11. Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry.
    Hilbert M, Erat MC, Hachet V, Guichard P, Blank ID, Flückiger I, Slater L, Lowe ED, Hatzopoulos GN, Steinmetz MO, Gönczy P, Vakonakis I.
    Proc Natl Acad Sci U S A. 2013, 110, 11373-8.
  12. Characterization of 14-3-3-ζ Interactions with Integrin Tails.
    Bonet R, Vakonakis I, Campbell ID.
    J Mol Biol. 2013, 425, 3060-72.
  13. Structural analysis of collagen type I interactions with human fibronectin reveals a cooperative binding mode.
    Erat MC, Sladek B, Campbell ID, Vakonakis I.
    J Biol Chem. 2013, 288, 17441-50.
  14. The GPS motif is a molecular switch for bimodal activities of adhesion class G protein-coupled receptors.
    Prömel S, Frickenhaus M, Hughes S, Mestek L, Staunton D, Woollard A, Vakonakis I, Schöneberg T, Schnabel R, Russ AP, Langenhan T.
    Cell Rep. 2012, 2, 321-31.
  15. Structural analysis of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope.
    Mayer C, Slater L, Erat MC, Konrat R, Vakonakis I.
    J Biol Chem. 2012, 287, 7182-9.
  16. Structural basis of the 9-fold symmetry of centrioles.
    Kitagawa D, Vakonakis I, Olieric N, Hilbert M, Keller D, Olieric V, Bortfeld M, Erat MC, Flückiger I, Gönczy P, Steinmetz MO.
    Cell. 2011, 144, 364-75.
  17. The streptococcal binding site in the gelatin-binding domain of fibronectin is consistent with a non-linear arrangement of modules.
    Atkin KE, Brentnall AS, Harris G, Bingham RJ, Erat MC, Millard CJ, Schwarz-Linek U, Staunton D, Vakonakis I, Campbell ID, Potts JR.
    J Biol Chem. 2010, 285, 36977-83.
  18. Implications for collagen binding from the crystallographic structure of fibronectin 6FnI1-2FnII7FnI.
    Erat MC, Schwarz-Linek U, Pickford AR, Farndale RW, Campbell ID, Vakonakis I.
    J Biol Chem. 2010, 285, 33764-70.
  19. Multi-factorial modulation of IGD motogenic potential in MSF (migration stimulating factor).
    Ellis IR, Jones SJ, Staunton D, Vakonakis I, Norman DG, Potts JR, Milner CM, Meenan NA, Raibaud S, Ohea G, Schor AM, Schor SL.
    Exp Cell Res. 2010, 316, 2465-76.
  20. Latrophilin signaling links anterior-posterior tissue polarity and oriented cell divisions in the C. elegans embryo.
    Langenhan T, Prömel S, Mestek L, Esmaeili B, Waller-Evans H, Hennig C, Kohara Y, Avery L, Vakonakis I, Schnabel R, Russ AP.
    Dev Cell. 2009, 17, 494-504.
  21. The structure of an integrin/talin complex reveals the basis of inside-out signal transduction.
    Anthis NJ, Wegener KL, Ye F, Kim C, Goult BT, Lowe ED, Vakonakis I, Bate N, Critchley DR, Ginsberg MH, Campbell ID.
    EMBO J. 2009, 28, 3623-32.
  22. Motogenic sites in human fibronectin are masked by long range interactions.
    Vakonakis I, Staunton D, Ellis IR, Sarkies P, Flanagan A, Schor AM, Schor SL, Campbell ID.
    J Biol Chem. 2009, 284, 15668-75.
  23. Identification and structural analysis of type I collagen sites in complex with fibronectin fragments.
    Erat MC, Slatter DA, Lowe ED, Millard CJ, Farndale RW, Campbell ID, Vakonakis I.
    Proc Natl Acad Sci U S A. 2009, 106, 4195-200.
  24. Structural analysis of the interactions between paxillin LD motifs and alpha-parvin.
    Lorenz S, Vakonakis I, Lowe ED, Campbell ID, Noble ME, Hoellerer MK.
    Structure. 2008, 16, 1521-31.
  25. Solution structure and sugar-binding mechanism of mouse latrophilin-1 RBL: a 7TM receptor-attached lectin-like domain.
    Vakonakis I, Langenhan T, Prömel S, Russ A, Campbell ID.
    Structure. 2008, 16, 944-53.

Link to all Vakonakis papers on PubMed

Dr. Ioannis Vakonakis
Associate Professor in Structural Biology and Biophysics

e-mail

Dr. Beatrice Li
Postdoctoral Researcher

e-mail

Dr. Natassa Kantsadi
Postdoctoral Researcher

e-mail

Jodie Ford
DPhil Student, Biochemistry Programme

 

Jonathan Machin
Part II Student

e-mail

 

Past Members

Dr. Michele Erat
Postdoctoral Research Associate, 2010-2013

Dr. George Hatzopoulos
Postdoctoral Research Associate, 2012-2015

Dr. Dirk Reiter
Postdoctoral Research Associate, 2013-2015

Ms. Leanne Slater
Laboratory Manager, 2010-2015

Dr. Christina Mayer
Dphil Student, 2008-2012

Dr. Kacper Rogala
Dphil Student, 2011-2015

Dr. Erin Cutts
Dphil Student, 2012-2016

Dr. Julia Busch
Dphil Student, 2013-2017

Dr. Jemma Day
Dphil Student, 2014-2018

Martin Masik
Part II Student

Alison Inglis
Part II Student

Dorothy Hawkins
Part II Student

Madeleine Butler
Part II Student

Samuel Bunt
Part II Student

Olivia Iller
Part II Student

Maria Kokolaki
Erasmus exchange student

Michael Chang
Part II (Princeton Exchange) Student

Niklas Laasch
MRes Student

Alex Carol
Undergraduate summer student

Raphael Trenker
Undergraduate Summer Student
Kerstin Feese
Undergraduate Summer Student
Jeong-Min (Thomas) Han
Dphil Rotation Student

Iris Blank
Undergraduate Summer Student

  Jonathan Shiroma
Undergraduate Summer Student

Group Photos

Jemma's viva, January 2019

Jemma's DPhil hat!

Julia's graduation, November 2018

With the group at New Forest National Park, summer 2018

Group Xmas outing, Bath, December 2017

Julia's DPhil viva party, November 2017

The most elaborate DPhil hat attempted yet (yes, this IS a hat!)

EMBO Centrosome and Spindle Pole Bodies conference - Heidelberg 2017

Centrosome meeting in Thun (Switzerland) - Summer 2017

'Why did they come to pester me?' Group handles Owls Day - Summer 2017

Xmas 2016 in Birmingham

Winners of the 2016 group archery competition!

Group summer day 2016

"Student with her poster" (Julia Busch - Astbury conversation 2016)

Xmas 2015

The Vakonakis' lab entry to the 2015 Great IDP Bake Off competition

Group summer day, 2015

BioMalPar, 2015 (with Peter Beck)

Summer group day, 2014

RSC UK NMR Discussion group, 2014

Christmas 2013

"Sinergia" centriolar initiative, 2013 (with Pierre Gönczy and Michel Steinmetz)

June 2012 (with Iain Campbell)

March 2011 (with Iain Campbell)

Christmas 2010 (with Iain Campbell)

June 2010 (with Iain Campbell and Jason Schnell)

Retirement symposium in honour of Prof. Iain Campbell, September 2009

Oxford LMB, 2007

Centriole components

C. elegans SAS-6 N-terminal domain and its variants

4G79

1.8 Å resolution model of a variant lacking the exposed a2-b5 loop

4GEX

2.8 Å resolution model of a stabilized N-N dimer (I154W, P3121 crystal form)

4GEU

2.65 Å resolution model of a stabilized N-N dimer (I154W, P21 crystal form)

3PYI

2.1 Å resolution model of the wild-type protein

 

Coiled-coil dimers of C. elegans SAS-6

4GFA

3.55 Å resolution model, P21212 crystal form

4GFC

2.85 Å resolution model, C2 crystal form

 

Structured domains of C. elegans SAS-5

4YNH

1.0 Å resolution model of the Implicodomain

4YV4

1.80 Å resolution model of the trimeric coiled coil

 

Zebrafish CPAP G-Box domain

4LD1

1.44 Å resolution model of G-box alone

4LZF

1.72 Å resolution model of G-box in complex with a short STIL peptide

4LD3

2.44 Å resolution model of G-box in complex with a long STIL peptide

 

 

 Malaria-related proteins

PfEMP1 intracellular segment (ATS)
 
P. falciparum PHIST protein PFI1780w
 
Human erythrocytic spectrin alpha chain repeats 16-17
 

2LKL

The structured "core" domain of ATS (ensemble of 39 NMR models)

4JLE

2.35 Å resolution model of the PFI1780w PHIST domain

5J4O

1.54 Å resolution model of the human spectrin repeats a16-17

 

 

 Other projects

Human fibronectin fragments 
 

3MQL

3.0 Å resolution model of 6FnI1-2FnII7FnI

2H41  2H45

2FnIII (averaged structure and ensemble of 25 NMR models)

2HA1

1-2FnIII (ensemble of 39 NMR models)


 

Human fibronectin in complex with collagen

   

3GXE

2.6 Å resolution model of 8-9FnI in complex with a low-affinity collagen a1(I) peptide

3EJH

2.1 Å resolution model of 8-9FnI in complex with a high-affinity collagen a1(I) peptide

 

Circadian clock protein KaiA
 

1M2E  1M2F

Pseudo-receiver domain of KaiA from Synechococcus elongatus (averaged structure and ensemble of 25 NMR models)

1Q6A  1Q6B

KaiC-interacting domain of KaiA from Thermosynechococcus elongatus (averaged structure and ensemble of 25 NMR models)

1SUY   1SV1

Thermosynechococcus elongatus C-terminal domain of KaiA in complex with a KaiC peptide (averaged structure and ensemble of 25 NMR models)

1R8J

2.03 Å resolution model of KaiA from Synechococcus elongatus

 

The rhamnose-binding domain of mouse Llatrophilin-1 GPCR

2JX9

RBL domain alone (ensemble of 25 NMR models)

2JXA

RBL domain in complex with rhamnose (ensemble of 25 NMR models)

 

Human 14-3-3-zeta

4HKC

2.2 Å resolution model of 14-3-3-zeta in complex with integrin alpha-4

 

Drosophila melanogaster RIM C2A domain

4TS6

1.92 Å resolution model

 

We are always looking for people with interest in malaria, centrioles or structural biology in general; informal enquiries from prospective graduate students and postdocs by e-mail(with CV) are welcome.

Funding for PhDs (called DPhils in Oxford) is provided through a number of different programmes. This page in the Departmental website provides a complete list. Students in our group are typically funded through the Wellcome Trust Structural BiologyBBSRC Interdisciplinary BioscienceEPSRC Systems Biology or Biochemistry programmes.

We advertise postdoctoral vacancies at the main Departmental website.

Vakonakis Lab

Department of Biochemistry
Rodney Porter building, 1st floor
University of Oxford
South Parks Road
Oxford OX1 3QU

Tel: +44 (0)1865 275725

Fax: +44(0)1865 613201

E-mail: ioannis.vakonakis@bioch.ox.ac.uk

Link to Directions and Maps

 

List of site pages