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Superrepression through Altered CorepressorActivated Protein:protein Interactions Chenlu He, Gregory S Custer, Jingheng Wang, Silvina Matysiak, and Dorothy Beckett Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01122 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 22, 2018

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Biochemistry

Superrepression  through  Altered  Corepressor-­Activated  Protein:protein   Interactions   Chenlu  He1,  Gregory  Custer2,  Jingheng  Wang1,  Silvina  Matysiak2  &  Dorothy  Beckett1*   Department  of  Chemistry  &  Biochemistry1,  Fischell  Department  of  Bioengineering2,   University  of  Maryland,  College  Park,  MD  20742       *Corresponding  author    

Contact:  email  [email protected]  

  Running  title:  Protein:protein  interactions  in  superrepression    

 

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Abstract    

Small  molecules  regulate  transcription  in  both  eukaryotes  and  prokaryotes  

by  either  enhancing  or  decreasing  assembly  of  transcription  regulatory  complexes.   For  allosteric  transcription  repressors  superrepressor  mutants  can  exhibit   increased  sensitivity  to  small  molecule  corepressors.  However,  since  many   transcription  regulatory  complexes  assemble  in  multiple  steps,  the  superrepressor   phenotype  can  reflect  changes  in  any  or  all  of  the  individual  assembly  steps.  E.  coli   biotin  operon  repression  complex  assembly,  which  responds  to  input  biotin   concentration,  occurs  via  three  coupled  equilibria  including  corepressor  binding,   holorepressor  dimerization  and  dimer  binding  to  DNA.  A  genetic  screen  has  yielded   superrepressor  mutants  that  repress  biotin  operon  transcription  in  vivo  at  biotin   concentrations  much  lower  than  required  by  the  wild  type  repressor.  In  this  work,   isothermal  titration  calorimetry  and  sedimentation  measurements  were  used  to   determine  the  superrepressor  biotin  binding  and  homodimerization  properties.  The   results  indicate  that,  although  all  variants  exhibit  biotin  binding  affinities  similar  to   that  measured  for  BirAwt,  five  of  the  six  superrepressors  show  altered   homodimerization  energetics.  Molecular  dynamics  simulations  suggest  that  the   altered  dimerization  results  from  perturbation  of  an  electrostatic  network  that   contributes  to  allosteric  activation  of  BirA  for  dimerization.  Modeling  of  the  multi-­‐ step  repression  complex  assembly  for  these  proteins  reveals  that  the  altered   sensitivity  of  the  transcription  response  to  biotin  concentration  is  readily  explained   solely  by  the  altered  superrepressor  homodimerization  energetics.  These  results   highlight  how  coupled  equilibria  enable  alterations  in  a  transcription  regulatory   response  to  input  signal  through  an  indirect  mechanism.     Key  words:  Transcription  repression,  protein:protein  interactions,  analytical   ultracentrifugation,  isothermal  titration  calorimetry,  All  Atom  MD  simulations  

 

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Biochemistry

  Introduction    

Communication  between  metabolism  and  transcription  regulation  provides  a  

mechanism  for  adjusting  gene  expression  in  response  to  an  organism's  demand  for   nutrients  and  metabolites.  In  bacteria  this  communication  is  frequently  achieved   through  binding  of  a  small  molecule  effector  to  a  transcription  regulatory  protein  to   allosterically  alter  its  sequence-­‐specific  binding  to  DNA.  In  negative  feedback   systems  such  as  the  bacterial  tryptophan  biosynthetic  pathway,  the  end  product  of   the  pathway,  tryptophan,  binds  to  the  repressor  to  promote  operator  binding,  thus   repressing  synthesis  of  the  biosynthetic  genes  when  metabolic  demand  for   tryptophan  is  satisfied  1.  For  an  inducible  system  exemplified  by  the  lactose  operon,   the  substrate  for  the  metabolic  pathway,  allolactose,  binds  to  the  lactose  repressor   to  relieve  repression,  thereby  allowing  synthesis  of  gene  products  required  for   lactose  transport  and  catabolism  2.  In  these  two  classic  systems  the  allosteric   effector,  corepressor  for  TrpR  and  inducer  for  LacI,  binds  to  a  repressor  oligomer  to   alter  its  affinity  for  DNA.  In  other  transcription  regulatory  systems,  effector  binding   can  change  DNA  occupancy  by  altering  the  regulatory  protein  oligomeric  state  3;  4.   Linkage  between  regulatory  protein  self-­‐association  and  effector  binding  yields  a   steep  response  of  transcription  level  to  small  changes  in  effector  concentration.      

The  bifunctional  E.  coli  biotin  repressor/ligase  links  biotin  utilization  to  its  

biosynthesis  by  functioning  as  both  an  enzyme  and  a  sequence  specific  DNA  binding   protein  5;  6.  In  its  enzymatic  function,  obligatorily  ordered  biotin  followed  by  ATP   binding  leads  to  biotinoyl-­‐5'-­‐adenylate  (bio-­‐5'-­‐AMP)  synthesis.  The  resulting  highly   stable  enzyme-­‐adenylate  complex,  holoBirA,  has  two  possible  fates  (Figure  17).  First,   it  can  bind  to  the  biotin  carboxyl  carrier  protein  (BCCP)  subunit  of  acetyl  CoA   carboxylase  to  catalyze  biotin  linkage  to  the  epsilon  amino  group  of  a  single  lysine   residue  on  the  protein.  This  post-­‐translational  biotin  addition  activates  carboxylase-­‐ catalyzed  synthesis  of  malonyl-­‐CoA,  the  substrate  in  fatty  acid  biosynthesis  5.   Alternatively,  holoBirA  can  homodimerize  and  bind  to  the  biotin  operator  sequence   to  regulate  transcription  of  the  biotin  biosynthetic  genes  (Figure  1,8;  9).      

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  Figure  1:  The  Biotin  Regulatory  System  :Biotin  binding,  followed  by  ATP,  results  in   bio-­‐5'-­‐AMP  synthesis  to  form  holoBirA.  HoloBirA  can  interact  with  apoBCCP  and   transfer  the  biotin.  Alternatively,  it  can  dimerize  and  bind  to  the  biotin  operator   sequence,  bioO,  to  repress  transcription  initiation.      

Partitioning  of  holoBirA  between  its  two  functions  is  regulated  by  the  

relative  rates  of  forming  the  alternative  protein:protein  interactions.  When  the   demand  for  biotin  is  relatively  high,  the  elevated  apoBCCP  concentration  favors   rapid  heterodimerization  between  holoBirA  and  the  acceptor  protein  10;  11;  12.  In   conditions  of  low  biotin  demand,  e.g.  slow  growth,  the  relatively  low  apoBCCP   concentration  allows  holoBirA  accumulation  and  relatively  slow  homodimerization.   The  resulting  homodimer  binds  to  the  biotin  operator  (bioO)  to  repress   transcription  initiation  at  the  biotin  biosynthetic  operon.  Thus  the  system  is  subject   to  kinetic  control  with  high  apoBCCP  concentrations  resulting  in  rapid  formation  of   the  heterodimeric  complex  between  BirA  and  the  carboxylase  subunit  and  low   acceptor  protein  concentrations  permitting  slow  homodimerization.      

In  addition  to  the  control  exerted  by  apoBCCP  concentration,  intracellular  

biotin  concentration  influences  BirA  function  13.  HoloBirA  dimerization  is  a   prerequisite  to  bioO  binding  and  repression.  Moreover,  bio-­‐5'-­‐AMP  binding  

 

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Biochemistry

regulates  the  homodimerization  energetics,  enhancing  it  by  -­‐4  kcal/mol  or  1000-­‐ fold  in  the  equilibrium  constant  14.  Since  adenylate  synthesis  occurs  by  an   obligatorily  ordered  mechanism,  with  biotin  binding  first,  biotin  concentration   regulates  repression  by  regulating  adenylate  synthesis  and  the  resulting  holoBirA   concentration  15.      

The  complex  set  of  inputs  into  the  biotin  regulatory  system  allows  for  several  

possible  levels  of  modulating  BirA  function.  Three  factors  must  be  considered  in   transcription  repression  complex  assembly  starting  from  apoBirA  (Figure  1  16).   First,  the  holoBirA  availability  is  limited  by  biotin  affinity  and  intracellular   concentration.  Second,  the  dimerization  free  energy  dictates  the  fraction  of  holoBirA   that  is  dimer,  the  active  species  in  bioO  binding.  Finally,  the  affinity  of  the  holoBirA   dimer  for  bioO  limits  the  fractional  saturation  of  the  operator  and  the  resulting  level   of  transcription  repression.  Perturbation  of  the  parameters  that  govern  any  of  these   three  steps  should  alter  the  transcriptional  response  to  biotin  concentration.      

Chakravartty  and  Cronan  recently  employed  a  genetic  screen  to  identify  BirA  

superrepressors  that  repress  transcription  at  the  biotin  operon  promoters  in  vivo  at   lower  biotin  concentrations  than  required  by  the  wild  type  protein17.  In  in  vitro   measurements  a  subset  of  the  purified  BirA  superrepressor  proteins  showed  higher   overall  affinity,  which  reflects  dimerization  plus  bioO  binding,  for  the  biotin   operator  sequence.  The  single  amino  acid  substitutions  that  yield  a  superrepressor   phenotype  are  located  in  the  central  or  catalytic/dimerization  domain  of  the  BirA   protein,  not  the  DNA  binding  domain.  Furthermore,  although  a  subset  of  the   substitutions  are  of  amino  acids  that  are  in  the  vicinity  of  the  dimerization  surface  in   the  holoBirAwt  structure,  these  amino  acids  do  not  directly  participate  in   noncovalent  interactions  at  the  dimer  interface  18.  Taking  into  account  the  three   inputs  into  the  transcription  regulatory  complex  assembly,  the  superrepressor   phenotype  could,  in  principle,  reflect  altered  biotin  binding,  dimerization  or   sequence-­‐specific  binding  of  the  resulting  holoBirA  dimer.  However,  previous   results  suggest  that  the  final  possibility  is  unlikely  since  holoBirA  variant  dimers    

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with  alanine  substitutions  on  the  dimerization  surface  all  bind  to  bioO  with  the   same  affinity,  despite  exhibiting  a  broad  range  of  self-­‐association  energetics  19.      

In  this  work  we  have  subjected  six  superrepressor  BirA  variants  to  biotin  

binding  and  homodimerization  measurements.  Isothermal  titration  calorimetry   (ITC)  measurements  reveal  that  all  variant  proteins  bind  to  biotin  with  affinities   similar  to  that  measured  for  BirAwt.    However,  sedimentation  equilibrium   measurements  indicate  that  the  bio-­‐5'-­‐AMP-­‐bound  proteins  are  altered  in   dimerization.  Results  of  molecular  dynamics  simulations  suggest  that  the  altered   dimerization  of  some  variants  reflects  changes  in  an  electrostatic  network  that  links   the  corepressor  binding  site  to  the  remainder  of  the  BirA  protein  structure.     Modeling  of  the  biotin  concentration  dependence  of  repression  complex  assembly   for  the  variants  indicates  that  altered  sensitivities  of  BirA  mutants  to  biotin  input   can  be  readily  explained  solely  by  altered  dimerization  properties.  These  results   illustrate  that  in  transcriptional  regulatory  systems  governed  by  linked  equilibria   the  response  to  input  can  be  significantly  changed  by  mutations  that  affect   protein:protein  interactions.  

 

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Biochemistry

Materials  and  Methods   Chemicals  and  Biochemicals   All  chemicals  and  biochemicals  were  at  least  reagent  grade.  Biotin  (Acros)  stock   solutions  were  prepared  in  Standard  Buffer  (10  mM  TrisHCl  [pH=7.5  at  20  °C],  200   mM  KCl,  2  mM  MgCl2)  and  stored  at  −80  °C.  Solutions  of  bio-­‐5′-­‐AMP,  which  was   synthesized  and  purified  as  previously  described3;  20,  were  prepared  in  Milli-­‐Q  H2O   and  stored  at  −80  °C  in  1ml  aliquots.  The  bio-­‐5’-­‐AMP  concentration  was  determined   by  UV  absorbance  at  259  nm  using  molar  extinction  coefficient  of  15,400  M-­‐1cm-­‐1.     Protein  Purification   The  overexpression  strains  for  BirA  suprerepressors  were  obtained  from  Dr.  John   Cronan,  University  of  Illinois.  The  recombinant  plasmids  are  pET19b  derivatives   that  encode  C-­‐terminal  hexahistidine-­‐tagged  BirA  variants  17.    The  E.  coli  strain   BL21(λDE3)  transformed  with  each  of  these  plasmids  was  grown  in  LB  media   containing  100  μg/ml  ampicillin.    When  the  culture  had  reached  an  OD600  of  0.8   protein  expression  was  induced  by  adding  isopropyl  β-­‐D-­‐1-­‐thiogalactopyranoside   (IPTG)  to  a  final  concentration  of  1  mM.  Induction  was  carried  out  for  4  hours  at   30°C  for  all  variant  except  G154D,  which  was  expressed  for  17  hours  at  20°C.  The   cells  were  disrupted  by  sonication  in  lysis  buffer  (100  mM  sodium  phosphate  [pH   6.5],  200  mM  NaCl,  5%  glycerol).  Polyethylene  Immine  (PEI)  was  added  to  the  lysis   supernatant  to  a  final  concentration  of  0.2%  to  precipitate  nucleic  acids.    After   centrifugation,  the  protein  in  the  resulting  supernatant  was  precipitated  by  adding   saturated  (NH4)2SO4  solution  to  a  final  concentration  of  60%  w/v.  Following   centrifugation  the  protein  pellet  was  resuspended  in  and  dialyzed  against  buffer   containing  50  mM  NaH2PO4  [pH  8.0],  300  mM  NaCl,  10  mM  imidazole,  5%  glycerol,   then  subjected  to  affinity  chromatography  on  Ni-­‐NTA  (Qiagen  &  Thermo  Scientific)  .   Non-­‐specifically  bound  proteins  were  removed  by  washing  the  column  with  the   same  buffer  containing  20  mM  imidazole.  The  protein  was  eluted  with  an  increasing   imidazole  concentration  (up  to  135  mM),  and  fractions  containing  BirA  were   dialyzed  against  buffer  containing  50  mM  TrisHCl  [pH  7.5  at  4oC],  50  mM  KCl,  5%   glycerol  and  0.1  mM  1,4-­‐dithiothreitol  (DTT).  Further  purification  was  carried  out    

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by  cation-­‐exchange  chromatography  on  SP  Sepharose  fast  flow  resin  (GE   Healthcare).    The  column  was  washed  with  the  above  buffer  and  protein  was  eluted   with  a  gradient  of  50-­‐375  mM  KCl.  For  G154D,  a  2nd  SP  Sepharose  column  was   required  using  a  gradient  of  50-­‐312.5  mM  KCl  to  elute  the  pure  protein.  The  purified   protein  was  dialyzed  against  storage  buffer  (50  mM  TrisHCl  [pH  7.5  at  4oC],  200  mM   KCl,  5%  glycerol,  and  0.1  mM  DTT)  and  stored  at  −80  °C  in  1  mL  aliquots.  The   protein  concentration  was  determined  by  UV  absorbance  at  280  nm  using  the   extinction  coefficient  of  47,510  M-­‐1cm-­‐1,  which  was  calculated  from  amino  acid   composition21.  All  variant  proteins  were  at  least  95%  pure  based  on  the  band   intensities  of  SDS-­‐PAGE  gel.       Isothermal  Titration  Calorimetry    

Biotin-­‐binding  measurements  were  carried  out  using  a  VP-­‐ITC  

microcalorimeter  (Microcal)  in  Standard  Buffer  (10  mM  TrisHCl  [pH=7.5  at  20  °C],   200  mM  KCl,  and  2.5  mM  MgCl2).  The  protein  was  dialyzed  against  Standard  Buffer   and  then  filtered  through  a  0.22  μm  PES  syringe  filter  (SIMSII)  to  remove  any   precipitate.  The  protein  concentration  in  the  resulting  sample  was  determined  by   UV  absorbance  at  280  nm.  The  dialysate  was  degassed  for  8  minutes  in  ThermoVac   (Microcal)  and  protein  and  ligand  were  both  gravimetrically  diluted  into  this  buffer   to  the  working  concentrations.  The  resulting  solutions  were  degassed  for  8  minutes   immediately  before  use.  For  all  measurements,  the  sample  cell  was  filled  with  the   protein  solution  at  a  concentration  of  2  μM  and  the  reference  cell  was  filled  with   Milli-­‐Q  H2O.  The  injection  syringe  contained  biotin  solution  at  a  concentration  of  20   μM.  All  titrations  were  performed  at  20  °C  with  23  injections  in  total,  with  a  2  μL   volume  for  the  first  injection  and  13  μL  for  the  remaining  22.  The  stirring  speed  was   310  rpm  for  all  titrations.      

Data  analysis  was  performed  in  Origin  7.0  (MicroCal,  Inc.).  Following  

integration  of  the  raw  titration  peaks,  the  ligand  injection  heat,  which  was  calculated   from  the  average  heat  of  last  five  injections,  was  subtracted  from  the  integrated  heat   associated  with  each  ligand  injection.  After  calculating  the  molar  enthalpy  per   injection  the  resulting  data  were  analyzed  using  a  single-­‐site  binding  model  to    

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obtain  the  stoichiometry,  n,  the  equilibrium  association  constant  KA,  and  the  molar   enthalpy  ΔH.  At  least  two  independent  biotin  titrations  were  performed  for  each   variant.     Sedimentation  Equilibrium    

The  homodimerization  constants  of  all  bio-­‐5’-­‐AMP-­‐bound  superrepressor  

proteins  were  determined  in  Standard  Buffer  by  sedimentation  equilibrium  using  an   Optima  XL-­‐1  analytical  ultracentrifuge  (Beckman  Coulter).  In  the  case  of  Y178C,   1mM  TCEP-­‐HCl  (PIERCE)  was  added  to  the  buffer  as  a  reducing  agent.  The  protein   was  dialyzed  exhaustively  against  Standard  Buffer  at  4°C  and  filtered  through  a  0.22   μm  PES  syringe  filter  (SIMSII)  to  remove  any  precipitation.  After  determining   protein  concentration  by  UV  absorption,  the  samples  were  prepared  at  three  protein   concentrations.  The  bio-­‐5’-­‐AMP  was  added  to  each  sample  at  a  1:1.5  protein:ligand   molar  ratio  under  stoichiometric  conditions.  For  all  measurements,  the  samples   were  centrifuged  at  20  °C  at  three  speeds  (18000,  21000  and  24000  rpm)  in   standard  12-­‐mm  six-­‐hole  cells  in  a  four-­‐hole  An-­‐60  rotor  (Beckman  Coulter).  After   eight  hours  at  each  speed,  absorbance  scans  were  acquired  at  290,  295,  or  300  nm   with  a  step  size  of  0.001  cm  and  five  replicates  per  step.  The  wavelength  was  chosen   based  on  the  total  protein  concentrations  in  the  samples  so  that  the  absorbance  falls   within  the  detection  limit  of  the  spectrophotometer.  Overlaying  of  scans  acquired  at   eight  and  nine  hours  of  centrifugation  at  each  speed  indicated  that  equilibrium  had   been  achieved.  At  least  three  independent  sedimentation  equilibrium   measurements  were  performed  for  each  variant.     Sedimentation  Velocity    

Sedimentation  velocity  measurements  were  performed  using  Optima  XL-­‐1  

analytical  ultracentrifuge  (Beckman  Coulter)  equipped  with  a  four-­‐hole  An-­‐60  rotor   (Beckman  Coulter).  The  protein  was  dialyzed  against  Standard  Buffer,  filtered   through  a  0.22  μm  PES  syringe  filter  (SIMSII)  to  remove  precipitate,  and  its   concentration  was  determined  by  uv  absorption.  The  resulting  protein  was  diluted   to  the  working  concentration  and,  if  appropriate,  bio-­‐5’-­‐AMP  was  added  to  the    

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sample  at  a  1:1.5  protein:ligand  molar  ratio.  Standard  two-­‐channel  charcoal-­‐filled   Epon  centerpieces  cells  with  a  3mm  or  12  mm  pathlength  were  used.  Prior  to   centrifugation  the  cells  and  rotor  were  equilibrated  at  20  °C  under  vacuum  for  at   least  2  hours.  The  centrifugation  was  run  at  20  °C  at  a  single  speed  of  50,000  rpm.   Continuous  scans  with  a  step  size  of  0.003  cm  and  one  replicate  were  acquired  at   290  nm.       Data  Analysis   Sedimentation  Equilibrium   The  absorbance  versus  radius  profiles  were  globally  analyzed  using  Nonlin22  in   HeteroAnalyisis  version  1.01.0060  (http://core.uconn.edu/resources/biophysics).   A  monomer-­‐dimer  model  was  used  to  obtain  the  best-­‐fit  equilibrium  dissociation   constant  governing  the  homodimerization  from  the  following  equation:    

 

c(r) = δ + c(ro

σ m (r 2 −ro2 ) 2 )e

1

2

+ c(ro ) (

K dim

2σ m (r 2 −ro2 ) 2   )e

(1)  

where  c(r)  is  the  protein  concentration  at  any  radial  position  ,  c(rO)  is  the  protein   concentration   at  reference  radial  position  rO,  δ  is  the  baseline  offset,  Kdim  is  the   € equilibrium  dissociation  constant  governing  dimerization  and  σm  is  the  reduced   molecular  weight  of  BirA  monomer,  which  was  obtained  using  the  following   relationship:    

 

 

σ=

(1− v ρ)ω 2     RT

 

 

where  M  is  the  molecular  weight  of  BirA  monomer,  

 

(2)  

 is  the  partial  specific  volume  

of  the  protein,   €ρ  the  buffer  density,  ω  is  the  angular  velocity  of  the  rotor,  R  is  the  gas   constant  and  T  is  absolute  temperature.  The  molecular  weight  of  His-­‐tagged  BirA  is   36,100  g/mol,  the  partial  specific  volume  of  0.755  ml/g  was  experimentally   determined8,  and  the  buffer  density  was  calculated  from  its  composition  at  the   working  temperature  using  Sednterp  (http://sednterp.unh.edu/).  In  all  analyses,   the  reduced  molecular  weight  of  the  dimer  was  assumed  to  be  twice  that  of  the   monomer.  The  quality  of  each  fit  was  assessed  by  the  magnitude  of  the  square  root   of  variance  and  the  distribution  of  the  residuals.      

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  Sedimentation  Velocity   Data  analysis  was  carried  out  using  DCDT+  version  2.4.3.2297623;  24.  A  subset  of   scans  (20-­‐30  scans)  was  used  to  produce  the  g(s*)  distribution.  The  data  were   analyzed  using  the  appropriate  model  to  obtain  the  sedimentation  coefficient  at   standard  condition  s(20,w).  The  partial  specific  volume  value  of  0.755  cm3/g  was   experimentally  determined8  and  the  solvent  density  of  1.008  g/ml  and  the  solvent   viscosity  of  1.002  cp  were  calculated  using  Sednterp  (http://sednterp.unh.edu/).     Molecular  Dynamics  (MD)  Simulations  and  Analysis    

MD  simulations  were  performed  for  wild  type  BirA  and  four  variants,  P143T,  

G154D,  Y178C,  and  M310L,  all  in  their  monomeric  form  in  complex  with  the   corepressor  analogue  biotinol-­‐5’-­‐AMP  (btnOH-­‐AMP).  Starting  coordinates  for  these   simulations  were  taken  from  chain  A  of  the  BirA  dimer  found  in  Protein  Data  Bank   (PDB)  entry  2EWN  18.  This  structure  contains  BirAwt  bound  to  btnOH-­‐AMP,  and  was   chosen  because  it  is  the  most  complete  of  the  available  BirAwt  structures,  having  all   residues  modeled  in  both  the  dimerization  and  ligand  binding  regions.  Amino  acid   substitutions  for  simulations  of  variants  were  made  in  PyMOL  25.  For  each   simulation  the  protein  model  was  placed  in  a  rhombic  dodecahedral  box  with  walls   extending  ~1nm  beyond  the  protein.  The  protein  was  then  solvated  with  ~20300   SPC/E  26  water  molecules.  Na+  counterions  were  added  to  the  system  as  needed  to   render  the  system  neutral,  replacing  randomly  chosen  water  molecules.  For  the  wt,   P143T,  Y178C,  and  M310L  variants  one  Na+  counterion  was  required,  while  for  the   G154D  variant  two  Na+  counterions  were  required.  The  energy  of  each  system  was   minimized  using  the  steepest  descent  method,  prior  to  production  runs.  Energy   minimization  was  then  followed  by  NVT  and  NPT  equilibration  runs  with  a  duration   of  100  ps  each,  using  position  restraints  with  a  force  constant  of  1000  kJ  mol-­‐1nm-­‐2   on  the  protein.  Production  runs  were  then  conducted  without  position  restraints,   using  an  NPT  ensemble  with  a  pressure  of  1  bar  and  a  temperature  of  300  K.   Production  runs  had  a  duration  of  1  μs,  with  the  last  500  ns  of  the  simulation  used  

 

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for  analysis,  as  we  have  shown  in  a  previous  publication  that  the  simulations  take  no   longer  than  500  ns  to  equilibrate  27.      

MD  simulations  were  performed  using  the  GROMACS  4.6  simulator  (28;  29;  30;  

31)  and  the  OPLS-­‐AA  force  field  32.  Parameters  for  btnOH-­‐AMP  were  kept  identical  to  

those  used  for  our  previous  publication,  and  are  available  upon  request  27.  The  time   step  for  simulations  was  set  to  2  fs,  with  neighbor  list  updates  occurring  every  five   steps.  Bond  lengths  were  constrained  using  the  LINCS  algorithm.  The    protein  and   water  temperatures  were  regulated  independently  using  the  V-­‐rescale  algorithm  33   with  a  time  constant  of  0.1  ps.  The  ligand  btnOH-­‐AMP  was  grouped  with  protein  for   temperature  coupling,  while  Na+  counterions  were  grouped  with  water.  Isotropic   pressure  coupling  was  used  in  all  simulations,  with  the  Parrinello-­‐Rahman  barostat   34,  using  a  time  constant  of  2  ps  and  a  compressibility  of  4.5  x  10-­‐5  bar-­‐1.    

   

Images  of  structures  from  MD  simulations  were  generated  using  VMD  (41).  

The  structures  represent  the  average  calculated  from  the  final  500  ns  of  each   trajectory  with  6,250  and  10,000  frames  used  for  the  wild  type  and  variants,   respectively.  These  average  structures  were  calculated  using  the  GROMACS  tool   g_covar  (33-­‐36).  Alignments  were  performed  in  VMD  (41)  using  the  backbone   alpha-­‐carbon  atoms.      

The  helicity  of  residues  140-­‐152  was  quantified  by  measuring  the  per-­‐

residue  average  RMSD  from  an  ideal  helix  (RMSDhx).  As  both  310-­‐  and  α-­‐helices   form  in  this  residue  range,  both  states  were  considered  in  this  calculation.  For  the   purposes  of  the  calculation,  ideal  helices  were  constructed  using  only  heavy   backbone  atoms  (N,  CA,  C,  and  O),  setting  the  φ  and  ψ  angles  to  -­‐57°  and  -­‐47°,   respectively,  for  the  310-­‐helix  and  to  -­‐49°  and  -­‐26°,  respectively,  for  the  α-­‐helix  35.  A   five-­‐residue  ideal  α-­‐helix  or  310-­‐helix  was  used  to  calculate  RMSDDα  or  RMSD310,   respectively.  For  each  residue,  i,  in  the  residue  range  140-­‐152,  these  ideal  helix   segments  were  aligned  to  a  five-­‐residue  segment  centered  on  residue  i,  and  RMSD   was  calculated  between  the  structures.  The  smaller  of  either  RMSDα  or  RMSD310   was  taken  as  the  RMSDhx  for  residue  i.  This  calculation  was  performed  for  each    

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Biochemistry

frame  in  the  final  500  ns  of  the  MD  simulation  trajectory,  with  the  average  per-­‐ residue  RMSDhx  reported.  

 

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Results   Biotin  binding  is  similar  for  all  superrepressors    

The  BirA  superrepressors  repress  transcription  in  vivo  at  lower  biotin  

concentrations  than  that  required  for  wtBirA.  A  potential  source  of  this  increased   sensitivity  is  an  increased  affinity  for  biotin,  which  was  investigated  by  measuring   the  biotin  affinities  of  the  variants  using  isothermal  titration  calorimetry  (ITC).  The   titration  data  for  the  M310L  variant  are  well-­‐described  by  a  single  site  model   (Figure  2A).  Moreover,  the  binding  parameters  resolved  from  the  data  analysis  are   similar  in  magnitude  to  those  measured  for  wtBirA  (Table  1).  

  Figure  2.  BirAM310L  function  in  biotin  binding  and  dimerization.  In  titration  of     BirAM310L  with  biotin  a    20µM  ligand  solutionwas  injected  into  a  2µM  protein   solution  (top).  The  resulting  binding  data  (squares)  were  analyzed  using  a  single   site  binding  model  (solid  line)  to  obtain  the  equilibrium  association  constant,  the   enthalpy  and  the  stoichiometry  of  binding.  B.  Sedimentation  equilibrium   measurement  of  the  monomer-­‐dimer  equilibrium  for  holoBirAM310L.  Data  from   samples  prepared  at  three  protein  concentrations  (left,  middle,  right),  and  acquired   at  three  rotor  speeds  (red,  blue,  black)  were  subjected  to  global  analysis  using  a  

 

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Biochemistry

monomer-­‐dimer  model  to  obtain  the  equilibrium  dissociation  constant.  The   residuals  of  the  fit  are  shown  in  the  bottom  panel.      

Biotin  binding  measurements  performed  on  the  remaining  superrepressors  

variants  indicate  that  they  bind  with  energetics  similar  to  those  measured  for   wtBirA.  The  quality  of  titration  data  obtained  for  these  variants  was  similar  to  that   shown  for  BirAM310L  (Figure  2A).  Within  error  the  equilibrium  constants  for  biotin   binding  differ  from  that  measured  for  the  wild  type  protein  by  less  than  2-­‐fold,  with   the  largest  difference  observed  for  BirAI187T.  The  binding  enthalpies  are  all  large  and   negative  with  the  P143T  variant  showing  the  largest  difference  of  2.6  kcal/mol  from   the  value  obtained  for  BirAwt  (Table  1).  Analysis  of  titrations  obtained  for  the   majority  of  the  variants  yielded  binding  stoichiometries  (n  values)  ranging  from  0.8-­‐ 1.0.    The  only  exception  is  BirAG154D,  for  which  the  relatively  low  value  of  0.73±0.02   may  be  related  to  instability.  Nevertheless,  this  variant  shows  binding  parameters   similar  to  those  measured  for  BirAwt.   Table 1: Biotin Binding Thermodynamics of BirA Superrepressor Proteins   Protein n Ka (M-1) ΔH (kcal/mol) -TΔS (kcal/mol) WT 0.84±0.07 2.4(±0.1)×107 -21.1±0.4 11.2±0.4 7 I187T 0.84±0.02 1.9(±0.1)×10 -19.5±0.4 9.8±0.4 K267M 0.83±0.03 2.7(±0.1)×107 -20.3±0.1 10.3±0.1 M310L 0.90±0.02 2.8(±0.3)×107 -20.2±0.1 10.23±0.04 P143T 0.867±0.004 2.4(±0.4)×107 -18.5±0.7 8.6±0.8 7 Y178C 0.84±0.04 2.4(±0.3)×10 -18.8±0.2 8.90±0.2 G154D 0.73±0.02 2.2(±0.3)×107 -20.2±0.3 10.3±0.4 o All measurements were conducted in Standard Buffer at 20 C. The errors represent the standard deviation resulting from averaging the results of at least three independent measurements.   Several  superrepressors  dimerize  more  tightly  than  holoBirAwt  

 

   

A  second  possible  source  of  the  enhanced  sensitivity  of  the  variants  to  biotin  

concentration  is  altered  dimerization  of  the  holo-­‐repressors.  This  was  tested  by   measuring  the  self-­‐association  of  the  holo-­‐variants  using  equilibrium  analytical   ultracentrifugation.  For  each  variant,  measurements  were  performed  on  samples  

 

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prepared  at  three  holo-­‐repressor  concentrations  that  were  centrifuged  at  three   rotor  speeds.  Global  nonlinear  least  squares  analysis  of  the  resulting  nine  data  sets   was  performed  using  both  a  single  species  and  a  monomer-­‐dimer  model.   Measurements  performed  on  holoBirAM310L  variant  indicate  that  it  dimerizes  with  a   Gibbs  free  energy  that  is  -­‐0.7±0.2  kcal/mol  more  favorable  than  does  holoBirAwt   (Figure  2B,  Table  2).     Table 2: Homodimerization Properties of BirA Superrepressor Proteins Protein Kdim, holo (M) ΔG°dim, holo (kcal/mol) s20,w (Svedbergs) -6 WT 11(±3)×10 -6.7±0.2 -6 I187T 3.4(±0.9)×10 -7.4±0.1 K267M 7(±2)×10-6 -7.0±0.2 M310L 3(±1)×10-6 -7.4±0.2 -6 P143T 3(±1)×10 -7.4±0.2 Y178C 7(±3)×10-4 -4.3±0.2 -8 G154D 7(±4)×10 -9.6±0.3 4.30±0.05 Measurements were conducted in Standard Buffer at 20oC and for each protein 9 data sets were globally analyzed using a monomer-dimer model. The errors represent the standard deviation resulting from averaging the results of at least three independent measurements.    

The  remaining  variants,  which  yielded  sedimentation  equilibrium  data  

similar  in  quality  to  that  obtained  for  holoBirAM310L,  exhibit  a  range  of  dimerization   energetics  (Table  2).  The  holoBirAP143T  and  holoBirAI187T  variants  dimerize  with  free   energies  similar  in  magnitude  to  that  measured  for  the  M310L  variant.  By  contrast,   holoBirAK267M  exhibits  dimerization  energetics  similar  to  those  of  wtBirA.  Finally,   the  holoBirAY178C  unexpectedly  dimerizes  with  a  Gibbs  free  energy  that  is   significantly  less  favorable  than  that  measured  for  holoBirAwt.      

The  G154D  variant,  which  shows  the  strongest  superrepressor  phenotype  in  

vivo,  also  exhibits  the  tightest  dimerization.  Indeed,  although  an  equilibrium   constant  for  holoBirAG154D  dimerization  of  7.1x10-­‐8  M  was  obtained  from  the   sedimentation  equilibrium  data  (Table  2),  the  species  populations  obtained  from  the   data  analysis  indicate  that  the  protein  is  nearly  100%  dimer.  Unfortunately,  the   detection  limits  of  the  absorption  optics  precluded  measurements  at  lower  protein   concentrations.  Results  of  sedimentation  velocity  measurements  performed  at   10µM  holoBirAG154D  indicate  that  90%  of  the  signal  is  associated  with  a  single    

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Biochemistry

species  characterized  by  a  sedimentation  coefficient  of  4.30±0.05S  (Table  2),   consistent  with  the  protein  being  primarily  in  the  dimer  state.  The  sedimentation   coefficient  obtained  for  the  holoBirAG154D  dimer  is  significantly  larger  than  the  3.9S   value  determined  for  holoBirAwt    dimer  12.  This  variant  has  previously  been  shown   to  migrate  in  SDS-­‐PAGE  at  an  apparently  higher  molecular  weight  than  do  BirAwt   and  other  variants  17  and  preparations  used  in  this  work  showed  the  same  aberrant   migration.        

Coupling  free  energies  for  two  of  the  variants  are  similar  to  that  measured  

for  the  wild  type  protein.  Corepressors  bio-­‐5'-­‐AMP  binding  to  BirAwt  enhances  its   dimerization  free  energy  by  -­‐4.0  kcal/mol,  which  reflects  a  1000-­‐fold  decrease  in  the   dimerization  equilibrium  dissociation  constant.  Although  four  of  the   superrepressors  dimerize  more  tightly  than  holoBirAwt,  it  is  not  known  if  these   proteins  also  dimerize  more  tightly  in  their  apo  forms.  In  other  words,  the   magnitude  of  the  coupling  between  effector  binding  and  dimerization  for  the   variants  is  not  known.  However,  assuming  a  coupling  free  energy  of  -­‐4.0  kcal/mol,  it   is  possible  to  calculate  the  dimerization  free  energies  of  the  apo  species  for  any   superrepressor.  For  example,  the  Gibbs  free  energy  of  holoBirAP143T  is  -­‐7.4  kcal/mol,   which  yields  an  expected  dimerization  free  energy  for  the  apo-­‐form  of  -­‐3.4  kcal/mol.   The  equilibrium  constant  calculated  from  this  free  energy  can  be  used  to  predict  the   fraction  dimer  as  a  function  of  total  protein  concentration.  These  calculations  yield   predictions  of  6%  and  4%  dimer,  respectively,  at  total  protein  concentrations  of  200   and  125uM  for  BirAP143T  and  BirAM310L  (Table  3).   Table  3.  Sedmentation  Velocity  Analysis  of  apoBirA  Variants   Predicted % Experimental Experimental BirA variant Predicted s20,w a dimer s20,w %dimerb WT (100µM) 1 4 2.732 2.762±0.003 P143T(200µM) 5 10 2.783 2.84±0.02 M310L(125µM) 4 6 2.762 2.79±0.02 a.  Prediction  is  based  on  the  assumption  that  the  coupling  free  energy  for  bio-­‐5'-­‐ AMP  binding  and  dimerization  is  -­‐4  kcal/mol  for  all  proteins.  b.  The  percent  dimer   was  calculated  from  the  experimentally-­‐determined  average  sedimentation   coefficient  assuming  that  it  represents  the  weight  average  of  contributions  from  the   monomer  and  dimer  species.    

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Sedimentation  velocity  provides  a  sensitive  method  to  detect  oligomeric  

species.  For  a  protein  that  rapidly  equilibrates  between  monomer  and  a  single   oligomer,  the  average  sedimentation  coefficient  should  deviate  from  that  of  the   monomer  and  increase  as  the  total  protein  concentration  is  increased36.   Sedimentation  velocity  measurements  were  performed  on  apoBirAP143T  and   apoBirAM310L,  at  total  concentrations  of  200  µM  and  125  µM,  respectively,  and  the   resulting  data  were  analyzed  to  identify  the  number  of  species  and  their   sedimentation  coefficients  (Table  3).  The  analysis  indicates  that  both  proteins  yield   a  single  peak  with  sedimentation  coefficients  that  are  in  reasonable  agreement  with   that  of  apoBirAwt  monomer.    The  absence  of  a  significant  amount  of  dimer  for  either   protein  at  the  concentrations  employed  indicates  that  the  coupling  free  energy  for   these  two  variants  is  at  least  as  favorable  as  the  -­‐4  kcal/mol  value  measured  for   BirAwt.     Perturbations  to  dimerization  reflect  altered  structures  of  the  holo-­variant   monomers    

Based  on  the  structure  of  the  holoBirA  dimer,  none  of  the  amino  acid  

residues  that  are  substituted  in  the  superrepressors  directly  participate  in  the  dimer   interface  in  the  wild  type  protein.  Consequently,  the  energetic  effects  of  the   substitutions  on  dimerization  are  difficult  to  rationalize.  This  is  particularly  true  for   the  superrepressors  with  substitutions  at  positions  far  from  the  dimer  interface   (Figure  3A).  In  the  absence  of  structural  data,  atomistic  simulations  were  used  to   probe  the  molecular  origins  of  these  energetic  changes.  Simulations  previously   performed  on  apo  and  holoBirAwt  monomers  indicate  good  agreement  with   experiment  27.  For  example,  the  structural  differences  between  the  apo  and  holo   species,  including  folding  of  the  biotin  binding  and  adenylate  binding  loop  (BBL,   ABL)  around  the  corepressor  bio-­‐5'-­‐AMP,  are  observed  in  simulations  (Figure  3B).   The  simulations  also  reproduce  the  disorder-­‐to-­‐order  transitions  that  the   dimerization  surface  loops  comprised  of  residues  140-­‐146  and  193-­‐199  undergo  as   well  as  the  interloop  packing.    Additionally,  the  conversion  of  residues  142-­‐145  

 

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from  coil  to  helix  to  extend  the  helix  formed  by  residues  146-­‐165  in  apoBirA  was   recapitulated  in  simulations.  Based  on  the  excellent  agreement  between  experiment   and  simulation  observed  for  the  wild  type  protein,  MD  simulations  were  used  to   investigate  the  molecular  origins  of  the  altered  dimerization  properties  of   superrepressor  variants.  

  Figure  3:  A.  Locations  of  single  amino  acid  substitution  in  the  holoBirA  dimer   structure  that  yield  the  superrepressor  phenotype.  Yellow:P143,  Pink:G154,  Orange:   Y178,  Cyan:I187,  Green:K267,  Blue:  M310.    B.  Apo  and  holoBirAwt  structures   illustrating  the  disorder-­‐to-­‐order  transitions  that  occur  on  the  ligand  binding  and   dimerization  surfaces  concomitant  with  bio-­‐5'-­‐AMP  binding.  Dashed  lines  in   apoBirA  indicate  disorder.  The  BBL  and  ABL  refer  to  the  biotin  and  adenylate   binding  loops,  respectively,  that  fold  around  bio-­‐5'-­‐AMP  in  holoBirA  C.  Root  mean  

 

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square  deviations  of  backbone  alpha  carbons  for  each  average  holoBirA  variant   from  that  of  the  average  holoBirAwt  structure.  Models  were  created  in  PyMol  25  using   pdb  files  1BIA  or  2EWN  as  input.      

Simulations  performed  on  the  superrepressor  variants  predict  that  the  single  

amino  acid  substitutions  yield  complex  changes  in  the  holoBirA  monomer  structure.   Computations  were  carried  out  on  four  of  the  variants  including  BirAP143T  and   BirAM310L  with  substitutions  located  on  the  dimerization  surface,  and  BirAG154D  and   BirAY178C  with  amino  acid  substitutions  in  the  central  domain  core.  The  simulations   were  performed  on  the  holo-­‐monomer  species  of  each  protein  for  a  total  of  1µs  and   analysis  was  performed  on  the  final  500  ns  of  each  trajectory,  at  which  time  each   simulation  had  reached  equilibrium.  An  overview  of  the  effects  of  each  amino  acid   substitution  on  the  protein  structure  was  first  obtained  by  calculating  the  root  mean   square  deviations  (RMSD)  of  the  C  backbone  atoms  for  the  average  structures  of   α

each  variant  relative  to  holoBirAwt  structure,  both  obtained  from  simulations   (Figure  3C).  Because  the  N-­‐terminal  DNA  binding  domain  has  previously  been   shown  to  exhibit  considerable  flexibility  both  internally  and  relative  to  the  central   domain,  simulation  results  for  only  the  central  and  carboxyl  terminal  domains  are   shown  18.  For  the  dimerization  surface  variants,  the  holoBirAP143T  structure  is   similar  to  that  of  the  wild  type  protein,  but  holoBirAM310L  variant  shows  large   structural  deviations  in  both  the  140-­‐146  and  193-­‐199  loops.  The  holoBirAG154D  and   holoBirAY178C  variants,  which  have  substitutions  in  the  central  domain  core,  are  both   characterized  by  large  RMSD  values  in  the  C-­‐terminal  domain.      

Structures  of  the  dimerization  loops  segments  observed  in  simulations  vary  

for  the  supperrepressors.  HoloBirAP143T  is  structurally  identical  to  holoBirAwt  with   respect  to  the  helical  conformation  of  residues  140-­‐152  as  well  as  the  interaction   between  the  140-­‐146  and  193-­‐199  loops  (Figures  3C,  4A).  By  contrast,  residues   142-­‐150  in  holoBirAM310L  deviate  significantly  from  their  structure  in  the  wild  type   protein  (Figure  4A).  However,  the  two  loops  are  more  closely  packed  in  this  variant  

 

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Biochemistry

than  they  are  in  the  wild  type  protein  (Figure  4B).  Like  the  P143T  variant,   holoBirAY178C  and  holoBirAG154D  show  dimerization  surface  loop  structures  identical   to  those  observed  in  the  wild  type  protein  (Figure  4A).    

  Figure  4.  A.  Average  per-­‐residue  RMSD  from  an  ideal  helix  for  residues  140-­‐152   calculated  from  simulations  performed  on  the  holoBirA  variants.  Error  bars  indicate   95%  confidence  intervals.    B.  Dimerization  surface  loops  140-­‐146  and  193-­‐199  for   holoBirAwt  (green)  and  holoBirAM310L  (blue).  *Results  for  BirAwt  were  originally   published  in  Wang,  et  al.  27.      

Comparison  of  the  experimentally  determined  apo  and  holoBirAwt  structures  

reveals  differences  in  the  C-­‐terminal  domain  that  may  contribute  to  allosteric   activation  of  dimerization.  The  comparison  reveals  an  electrostatic  network  in   holoBirAwt  that  incorporates  both  central  and  C-­‐terminal  domain  residues  (Figure   5).  Although  a  subset  of  these  interactions  is  present  in  apoBirAwt,  a  number  of  them   can  form  only  in  the  holo-­‐protein  because  BBL  residues  R118,  R119  and  R121,   which  are  disordered  in  the  absence  of  ligand,  participate  in  the  network.   Importantly,  the  average  holoBirAwt  structure  obtained  from  molecular  dynamics   simulations  reveals  a  network  that  is  nearly  identical  to  that  observed  in  the   experimentally  determined  structure  (Figure  5).  

 

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  Figure  5.  Electrostatic  networks  in  BirA  variants:  Network  shown  on  the  holoBirA   structure  (upper  left)  and  cartoon  representations  of  the  network  in  BirAwt  and   super  repressor  variants.  The  positively  charged  residues  are  shown  in  blue,   negatively  charged  in  read  and  neutral  polar  residues  in  grey.  The  model  was   created  in  PyMol25  using  2EWN  as  the  input  file.      

The  electrostatic  network  is  altered  in  the  simulated  holoBirA  variant  

structures.  The  network  in  the  holoBirAP143A  variant  shows  a  loss  of  two  interactions   and  a  gain  of  an  interaction  between  R119  and  E313  (Figure  5).  The  holoBirAM310L   variant  network  is  nearly  identical  to  that  of  holoBirAwt,  with  loss  of  only  one   charged  hydrogen  bond  between  Y178  and  K272.  For  the  holoBirAG154D  variant  the   insertion  of  the  additional  charged  residue  is  accompanied  by  both  disruption  of  

 

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some  interactions  and  addition  of  others.  By  contrast,  the  holoBirAY178C  variant   shows  loss  of  multiple  electrostatic  interactions  that  are  present  in  holoBirAwt.  The   differences  in  the  network  observed  for  holoBirAG154D  and  holoBirAY178C  correlate   with  changes  in  the  orientation  of  C-­‐terminal  domain  relative  to  the  central  domain   (Figure  6).  In  each  panel  the  C-­‐terminal  domain  alignment  was  obtained  from   pairwise  alignment  of  the  central  domain  backbone  C  atoms  of  the  relevant   α

holoBirAvariant  with  those  of  holoBirAwt.  Notably,  the  backbone  C  alignments  of  the   α

isolated  C-­‐terminal  domain  of  each  variant  with  that  of  the  wild  type  protein   indicate  close  overall  agreement.  Thus  the  differences  observed  in  Figure  6  reflect   differences  in  the  C-­‐terminal  domain  orientation  relative  to  the  central  domain.    

  Figure  6:  C-­‐terminal  domain  structures  are  altered  in  some  superrepressor  variants.   Alignments  of  holoBirAwt:(grey)  C-­‐terminal  domain  with  that  of  (A.  )  holoBirAP143T,     (B.)  holoBirAM310L,  (C.)  holoBirAG154D,  and  (D)  holoBirAY178C.  Alignment  of  the  central  

 

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domain  (residues  80  to  269)  of  the  average  structure  of  each  variant  with  that  of   holoBirAwt  was  performed  using  MultiSeq  in  VMD37.  

 

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Discussion      

The  BirA  superrepressors  repress  transcription  in  vivo  at  biotin  

concentrations  lower  than  those  required  by  BirAwt.  Nevertheless,  ITC   measurements  indicate  that  all  of  the  proteins  have  biotin  binding  properties   similar  to  those  measured  for  BirAwt.    It  is  possible  that  the  proteins  have  an   increased  affinity  for  bio-­‐5'-­‐AMP.  However,  their  tight  dimerization  precludes   measurement  of  adenylate  binding  because  the  ligand  binding  cannot  be   deconvoluted  from  the  coupled  dimerization  process.  The  biotin  binding  data   indicate  that  altered  sensitivity  to  a  transcription  regulatory  system  input  can  be   achieved  in  the  absence  of  any  change  in  repressor  affinity  for  the  input  ligand.      

With  a  few  notable  exceptions  the  superrepressor  dimerization  free  energies  

agree  with  the  results  reported  by  Chakravartty  and  Cronan  17.  First,  consistent  with   results  of  in  vivo  gene  expression  measurements,  the  G154D  variant  shows  the   tightest  dimerization  (Table  4).  Although  the  P143T,  M310L  and  I187T  variants  all   dimerize  with  identical  Gibbs  free  energies,  BirAI187T  exhibits  slightly  greater   repression  of  β-­‐galactosidase  expression  in  bioF::lacZ  fusion  strains.  The  K267M   variant,  which  exhibits  a  superrepressor  phenotype  in  vivo,  dimerizes  with  a  Gibbs   free  energy  identical,  within  error,  to  that  of  the  wild  type  protein.  Finally,  despite   displaying  a  superrepressor  phenotype  similar  to  that  of  the  M310L,  P143T  and   I187T  variants,  the  Y178C  variant  dimerizes  much  more  weakly  than  BirAwt.  Similar   results  were  obtained  on  multiple  preparations  of  the  protein  in  the  absence  and   presence  of  the  reducing  agent  TCEP.  Moreover,  the  sequence  of  the  entire  coding   sequence  for  the  variant  in  the  expression  plasmid  was  verified.  Thus,  the  origin  of   the  discrepancy  between  in  vivo  and  in  vitro  measurements  for  this  protein  is   currently  unknown.  

 

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    Table 4: Comparison of in vivo and in vitro properties of BirA superrepressors   Biotinylation bioF::lacZY bioF::lacZY ΔG°biotin ΔG°dim, holo activitiesc, Variant a b expression derepression kcat/Km (kcal/mol) (kcal/mol) (M-1s-1) wt 17.3 7.36 1.6±0.3x105 -9.90±0.02 -6.7±0.2 I187T 1.6 3.04 1.16±0.02x105 -9.76±0.03 -7.4±0.4 K267M 5.4 4.14 + -9.97±0.02 -7.0±0.2 5 M310L 4.9 2.61 1.4±0.1x10 -9.98±0.03 -7.6±0.4 P143T 4.2 2.24 + -9.90±0.03 -7.6±0.4 Y178C 4.1 3.04 + -9.90±0.03 -4.5±0.4 G154D 0.3 0.86 n.d. -9.84±0.04 -9.6±0.3 a

The bioF::lacZY expression was determined using β-galactosidase activity assays in cell culture containing 1.6nM biotin17. b Fold bioF::lacZY derepression in response to overexpression of the biotin acceptor protein (AccB-AccC) measured in 40nM biotin. c The symbol + represents the ability to ligate biotin to AccB-87 in vitro, whereas n.d. represents no detectable biotin transfer.    

Given  the  complexity  of  the  E.  coli  Biotin  Regulatory  System,  multiple  

potential  sources  of  the  observed  discrepancy  between  the  in  vivo  and  in  vitro   results  obtained  for  the  BirAY178C  variant  were  considered.    For  example,  this  variant   is  altered  in  its  competitive  interaction  with  apoBCCP  in  biotin  transfer.  However,  in   vivo  measurements  of  derepression  resulting  from  BCCP  overexpression  and  in  vitro   measurements  of  biotin  transfer  indicate  that,  with  the  exception  of  the  G154D   variant,  all  superrepressors  are  similar  to  BirAwt  in  their  biotin  transfer  properties   (Table  4)  17.  Moreover,  in  the  strain  used  for  identifying  superrepressor  mutants  the   biotin  transfer  function  was  supplied  by  a  plasmid  born  heterologous  yeast  biotin   protein  ligase17,  which  rendered  the  superrepressor  phenotype  independent  of   biotin  transfer  function.  A  second  potential  source  of  the  discrepancy  is  that  the   affinity  of  the  holoBirAY178C  dimer  for  bioO  is  significantly  higher  than  that  of  the   holoBirAwt  dimer.  However,  previous  studies  reveal  that  the  energetics  of  the   holoBirA  dimer:bioO  binding  interaction  is  independent  of  dimerization  free   energy19.    A  final  consideration  that  was  not  addressed  in  in  vivo  studies  of  the  

 

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superrepressors  is  the  intracellular  concentrations  of  the  variant  proteins.  However,   overexpression  of  the  BirAY178C  variant  for  purification  revealed  no  difference  in  its   levels  relative  to  those  observed  for  the  wild  type  protein.      

The  holoBirAwt  structure  combined  with  results  of  MD  simulations  provides  

predictions  of  the  molecular  origins  of  the  altered  superrepressor  dimerization   properties.  In  BirAP143T  and  BirAM310L,  the  substituted  residues  are  located  in   dimerization  surface  loops  (Figure  3A).  The  P143  residue  participates  in  the  coil  to   helix  transition  that  residues  142-­‐145  undergo  upon  bio-­‐5'-­‐AMP  binding.  Previous   studies  of  the  holoBirAP143A  variant,  which  dimerizes  less  tightly  than  does   holoBirAwt,  indicate  that  it  does  not  undergo  this  transition27.  Consistent  with  its   relatively  strong  dimerization,  simulations  performed  on  holoBirAP143T  indicate   preservation  of  the  helical  structure.    The  M310L  substitution,  which  enhances   dimerization,  is  located  in  the  BirA  C-­‐terminal  domain  and  simulations  suggest  that   this  substitution  alters  dimerization  through  enhancing  the  packing  between  the   140-­‐146  and  193-­‐199  loops  on  the  dimerization  surface  (Figure  4B).      

In  the  G154D  and  Y178C  variants  with  substitutions  in  the  central  domain  

core,  shifts  in  the  relative  orientation  of  the  central  and  C-­‐terminal  domains  may  be   responsible  for  altering  BirA  dimerization.  Bio-­‐5'-­‐AMP  binding  to  BirAwt  is   accompanied  by  formation  of  an  electrostatic  network  involving  BBL  residues  R118,   R119  and  R121,  which  are  disordered  in  the  absence  of  ligand18;  38.    Comparison  of   the  apo  and  holoBirAwt  structures  indicates  that  bio-­‐5'-­‐AMP  binding  is  accompanied   by  rotation  of  the  C-­‐terminal  domain  relative  to  the  central  domain  18.  Since  the   electrostatic  network  physically  connects  the  BBL  to  the  C-­‐terminal  domain,  this   rotation  is  likely  associated  with  network  formation.  Simulations  predict  that  the   electrostatic  network  is  altered  in  both  holoBirAG154D  and  holoBirAY178C.  Moreover,   although  backbone  C  alignments  indicate  that  the  structure  of  the  domain  itself  is   α

similar  for  the  wild  type  and  variant  proteins,  BirAG154D  and  BirAY178C  show  a  shift  in   the  orientation  of  the  C-­‐terminal  domain  relative  to  the  central  domain  (Figure  6).    

 

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Future  structural  studies  will  provide  tests  of  this  predicted  coupling  between   electrostatic  network  formation  and  the  domain  shifts  in  BirA.      

Modeling  of  the  biotin  concentration  dependence  of  transcription  repression  

complex  assembly  reveals  that  it  is  highly  sensitive  to  holoBirA  dimerization   energetics.  As  indicated  in  the  Introduction  (Figure  1),  biotin  operon  transcription   repression  complex  assembly  occurs  in  multiple  steps.  First,  biotin  followed  by  ATP   binding  results  in  bio-­‐5'-­‐AMP  synthesis  and  accumulation  of  holoBirA  monomer.   This  obligatorily  ordered  mechanism  renders  the  fractional  saturation  of  BirA  by   bio-­‐5'-­‐AMP  dependent  only  on  biotin  binding.  Therefore,  holoBirA  concentration   can  be  predicted  using  the  following  equation:     [holoBirA] = [BirA]total Ybiotin = [BirA]total

K A,biotin [biotin]  (3)   1+ K A,biotin [biotin]

in  which  [holoBirA]  is  the  BirA•bio-­‐5’-­‐AMP  concentration,  [BirA]total  is  the  total  BirA   €concentration,   Y biotin  is  the  fractional  saturation  of  BirA  with  biotin,  KA,biotin    is  the  

equilibrium  association  constant  governing  biotin  binding  to  BirA  and  [biotin]  is  the   free  biotin  concentration.  HoloBirA  assembly  onto  the  biotin  operator  sequence,   € bioO,  occurs  through  coupled  dimerization  and  holoBirA  dimer  binding  to  bioO.   Thus,  the  fractional  saturation  of  bioO  by  holoBirA,  

,  is  expressed  by  the  

following  equation:      

YbioO =

K DIM K bioO [holoBirA]2 1 +K DIM K bioO [holoBirA]2

 

 

 

(4)  

  €in  which  KDIM  and  KbioO  are  the  equilibrium  association  constants  governing  

holoBirA  dimerization  and  dimer  binding  to  bioO,  respectively.       Combined  Equations  (3)  and  (4)  allow  predictions  of  the  dependence  of   biotin  operator  occupancy  on  biotin  concentration  at  a  single  total  BirA   concentration16.  These  predicted  curves  have  previously  been  shown  to  agree   reasonably  well  with  in  vivo  measurements  of  the  dependence  of  transcription  level  

 

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on  biotin  concentration16.  Predicted  fractional  saturation  curves  for  four  BirA   variants  are  shown  in  Figure  7.  The  KA,biotin  and  KDIM  values  used  in  the  simulations   were  obtained  from  the  ITC  and  sedimentation  equilibrium  measurements,   respectively,  reported  in  this  work.  The  magnitude  of  the  KbioO  parameter  used  for   all  variants  was  the  value  obtained  for  holoBirAwt  binding  using  DNaseI  footprint   titration19.  The  total  BirA  concentration  of  6.6x10-­‐7M  was  calculated  from  the   absolute  protein  synthesis  rate  obtained  by  ribosome-­‐profiling  39   (http://ecoliwiki.net/tools/proteome/)  and  cell  volume  for  E.  coli  estimated  as  1.3   µm3  (http://book.bionumbers.org/how-­‐big-­‐is-­‐an-­‐e-­‐coli-­‐cell-­‐and-­‐what-­‐is-­‐its-­‐mass/).  

  Figure  7:  Modeling  sensitivity  of  the  dependence  of  bioO  fractional  saturation  by   BirA  on  biotin  concentration  for  BirAG154D:Pink:,  BirAM3!0L  Blue,  BirAwt:Black;   BirAY178C:Orange.  See  text  for  a  description  of  the  equations  and  parameters  used  in   generating  the  curves.     The  steep  dependence  of  bioO  saturation  on  biotin  concentration  observed  in   all  curves  reflects  the  coupling  between  biotin  binding,  which  triggers  bio-­‐5'-­‐AMP   synthesis,  dimerization  and  DNA  binding  in  repression  complex  assembly  (Figure  

 

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7).  The  tighter  dimerizing  protein,  BirAM310L,  occupies  the  operator  at  lower  biotin   concentration  than  does  BirAwt.  BioO  saturation  by  the  G154D  variant  is  sensitive  to   even  lower  biotin  concentration,  with  a  midpoint  or  0.5  fractional  saturation   occurring  at  nanomolar  biotin  concentration.  By  contrast,  the  weakly  dimerizing   Y178C  variant  requires  significantly  higher  biotin  concentration  to  achieve  bioO   occupancy.  Thus,  the  altered  dimerization  energetics  of  the  variants  are  predicted  to   result  in  a  two  orders  of  magnitude  range  of  sensitivities  of  occupancy/repression   to  biotin  concentration.  Direct  comparison  of  the  predicted  dependence  of  bioO   occupancy  on  biotin  concentration  will  require  detailed  in  vivo  measurements.16      

In  general  several  factors  can  contribute  to  the  sensitivity  of  any  

transcriptional  response  to  the  input  stimulus.  Two  levels  of  sensitivity  to  input  can   be  considered.  The  first  refers  to  the  concentration  span  of  input  molecule  over   which  the  response  occurs,  with  a  narrow  span  indicating  high  sensitivity  to   changes  in  input  concentration.  Coupled  reactions  are  typically  responsible  for  a   steep  response.  For  example,  in  the  bacteriophage  lambda  lysogeny  to  lysis  switch   the  steepness  of  the  transcription  response  to  changing  input  cI  repressor   concentration  reflects  coupling  between  protein:protein  and  protein:DNA   interactions  40.  The  steepness  or  cooperativity  of  the  transcription  response  to   tetracycline  concentration  mediated  by  the  tetracycline  repressor  is  correlated  with   the  arrangement  and  juxtaposition  of  regulatory  sites  as  well  as  the  promoter   sequences  associated  with  a  particular  regulon  41.  For  the  biotin  biosynthetic  operon   the  steep  transcription  response  to  biotin  concentration  reflects  the  three-­‐coupled   equilibria  that  dictate  the  fractional  occupancy  of  the  bioO  DNA  sequence  by   holoBirA.  A  second  measure  of  sensitivity  is  the  minimum  amount  of  input  signal   required  to  achieve  a  response.  Altering  the  affinity  of  the  regulatory  protein  for  the   input  signal  molecule  biotin  provides  a  direct  route  to  changing  this  sensitivity.  The   results  provided  in  this  work  indicate  that  a  broad  range  of  sensitivities  of  a   transcriptional  response  to  input  signal  concentration  can  also  be  achieved  by   modulating  the  self-­‐association  energetics  of  the  transcription  regulatory  protein.   Further,  the  BirA  dimerization  free  energy  is  subject  to  significant  changes  resulting    

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from  single  amino  acid  substitutions  at  amino  acid  positions  that  are  distributed   throughout  the  protein  structure.

 

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Acknowledgements: This work was supported in part by NIH Grant S10 RR15899 to DB. The authors thank Dr. John E. Cronan for the plasmids used for protein expression.   References   1.  

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