# Two Distantly Spaced Basic Patches in the Flexible Domain of Huntingtin-Interacting Protein 1 (HIP1) Are Essential for the Binding of Clathrin Light Chain

Authors: Joel A. Ybe; Mary E. Clegg; Melissa Illingworth; Claire Gonzalez; Qian Niu
Journal: Research Letters in Biochemistry
 (2009)

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## Abstract

The interaction between HIP family proteins (HIP1 and HIP12/1R) and clathrin is fundamental to endocytosis. We used circular dichroism (CD) to study the stability of an HIP1 subfragment (aa468-530) that is splayed open. CD thermal melts show HIP1 468-530 is only stable at low temperatures, but this HIP1 fragment contains a structural unit that does not melt out even at 83C∘. We then created HIP1 mutants to probe our hypothesis that a short hydrophobic path in the opened region is the binding site for clathrin light chain. We found that the binding of hub/LCb was sensitive to mutating two distantly separated basic residues (K474 and K494). The basic patches marked by K474 and K494 are conserved in HIP12/1R. The lack of conservation in sla2p (S. cerevisiae), HIP1 from D. melanogaster, and HIP1 homolog ZK370.3 from C. elegans implies the binding of HIP1 and HIP1 homologs to clathrin light chain may be different in these organisms.


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## Body

## 1. Introduction

Huntingtin-interacting protein 1 (HIP1) and
its relative, HIP12/1R, contribute to the budding of clathrin-coated vesicles
(CCVs) [1, 2].  The yeast homolog of
HIP1, sla2p, is required for the
development of CCVs in yeast [3, 4].
There are shared regions in HIP1 and HIP12/1R that mediate the binding
of clathrin, but there are also sites that are unique to each HIP protein.  For example, HIP1, but not HIP12/1R, has a
clathrin box motif (LMDMD332) which functions to bind the N-terminal beta
propeller domain of clathrin [5].  HIP1
and HIP12/1R do not interact with adaptor protein 2 (AP2) in the same way.  The AP2-binding FXDXF and DPF motifs (X
denotes any amino acid) are only in HIP1 [5].
HIP12/1R apparently has a higher affinity for F-actin [6], suggesting
that HIP1 and HIP12/1R play distinct roles in the formation of CCVs.  A number of amino acids (L486 and
R487 in the DLL486R487KN region) in the
coiled-coil domain of HIP1 and HIP12/1R have been identified to impact the
binding of clathrin light chain (CLC) [7], but we do not know if L486 and R487 constitute the entire CLC binding determinants in HIP
proteins.  We recently determined the
crystal structures of two contiguous subfragments of the HIP1 coiled-coil
domain (PDB files: 2NO2 and 2QA7) that span the clathrin light chain-binding
region [8, 9].  The DLL486R487KN
stretch was embedded in the splayed opened region that we first discovered in
HIP1 482-586 (2NO2) [8] and later found in HIP1 371-481 (2QA7) [9].  In this research letter we define the
inherent stability of a segment of HIP1 that contains the opened region and ask
if electrostatic interactions help drive the binding of CLC.  We generated an HIP1 subfragment (aa468-530) that spans the
opened region for circular dichroism (CD) experiments to assess this domain’s
intrinsic stability.  We then studied if
two distantly spaced positively charged patches in the opened region played any
role in the binding of CLC.  Here we
report that the HIP1 468-530 construct is unstable, but paradoxically a
heat-resistant structural unit is present within this subfragment.  We found that the two basic patches in the
flexible part of HIP1 (centered on K474 and K494) are crucial for the binding
of CLC.  These electrostatic determinants
are part of a solvent exposed hydrophobic surface that we previously argued was
suitable for CLC.


## 2. Results and Discussion

### 2.1. Coiled-Coil Segment of HIP1 that Includes Determinants for Clathrin Light Chain Is Intrinsically Unstable

The binding of CLC to HIP1 requires L486 or R487 [7] (human
HIP1 numbering (accession number NP 005329), conserved in HIP12/1R).  We recently located L486 and R487 to a
flexible segment of HIP1 (position 2, Figure 1), in S3 path [8].  Here we used CD to probe the stability of
HIP1 468-530 (marked by the grey strip in Figure 1, between Y468 and R547), a
coiled-coil segment that has the S3 path.
At 4°C the CD profile was helical, indicated by the double minimum at
208 and 222 nm (Figure 2).  The ratio of
intensities at 222 and 208 nm can be used to distinguish coiled coils from
isolated helices (≥1 for coiled coils and <0.86 for
isolated helices [10, 11]).  The ratio
values in Figure 2 suggest that HIP1 468-530 shifts from a mixture of isolated
helices and coiled coils at low temperatures to isolated helices at 37°C.  We point out
that the 37°C profile is almost identical to the 83°C scan, suggesting that HIP1
468-530 contains a heat-stable element. We are presently doing NMR experiments to
map out the extent of the heat unstable portion of HIP1 468-530.Figure 1
Structural features of the HIP1 clathrin
light chain-binding site.  Two distantly
separated basic amino acids in the opened region of HIP1 mediate the binding of
clathrin light chain.  The assembled
model was generated using PDB files 2NO2 [8] and 2QA7 [9].  For the sake of clarity, we only show a
portion of the second HIP1 helix (dark grey) that stops before Y468. The yellow
bar marks the position of the solvent exposed hydrophobic S3 path [8].  The new data in Figure 3 indicate that K474
is a strong determinant for binding and imply that S3 path begins before the DLL486R487KN
region.  The N- and C-termini of the HIP1
crystal structure are labeled N and C.
The numbers 1–3 along the
yellow bar mark the position of amino acids that control the binding of
clathrin light chain (position 1: K474; position 2: L486 and R487 reported by
the McPherson group [7]; and position 3: K494).
K474 and K494 are ~50 Å apart.
R500, R508 or K511 (see arrow) do not participate in binding and
therefore define the boundary of the light chain-binding site.  The HIP1 468-530 subfragment
used in the CD studies in Figure 2 spans across an opened region of the HIP1
coiled coil in our 2NO2 and 2QA7 crystal structures.  The HIP1 model was created using PyMol
(http://www.pymol.org).Figure 2
HIP1 468-530 subfragment in the opened region is stable only at low
temperature.  Equilibrium far UV CD scans
were performed at4°C
(open triangle), 10°C (open diamond), 37°C (cross), and 83°C (open
circle).  The position of the 222 nm
signal did not change with temperature, but the 208 nm signal shifted as the
sample was heated.  The 222/208 nm ratios
were calculated from the raw CD data.
Because the signal below 200 nm was noisy, we did not attempt to get the
helix content from the CD data.


### 2.2. Basic Patches Centered on K474 and K494 in HIP1 Are Essential for Binding Clathrin Light Chain

We
investigated if a series of basic patches in the opened region could
participate in binding CLC.  K494 (see position
3, Figure1) is in S3 path (yellow bar, Figure 1) previously described [8] and
is followed by a cluster of basic residues (indicated by the arrow in Figure
1).  K474 (position 1) is located before
the DLL486R487KN region, close to the part of HIP1 that
mediates the binding of Huntingtin interacting protein 1 interactor (HIPPI)
[9].  To probe if those in S3 path contributed, we made 5 GST-HIP1h
(HIP1h is aa370-644) 370-644 mutants (K494A, K494E, R500E, R508E, and
K511E).  We performed GST pulldowns to
evaluate the binding of 6Hishub/6HisLCb (hub is central third of clathrin
(aa1074-1675) [12] and 6HisLCb is N-terminally histidine-tagged bovine clathrin
light chain b with the neuronal insert).
It is important to study 6HisLCb that is bound to 6Hishub to closely
mimic how HIP1 interacts with clathrin baskets in cells.  Every GST pulldown was done at least three
times, using freshly isolated proteins and charged GST beads each time.  Clathrin hub (N-terminally histidine tagged)
was detected by western blotting with a commercial histidine tag antibody.  LCb was blotted with CON.1 monoclonal
antibody and GST-HIP1h constructs were visualized with a commercial GST
antibody.  The anti-GST bands in Figures 3(a) and 3(b) showed that
the GST-HIP1h levels were balanced (loading control).  The negative controls in lanes 1–3 in Figure 3(a)
and lanes 1–3 in Figure 3(b)
show that the GST signals from each binding experiment were not random
interactions, but reflected true binding events.  The level of 6Hishub/6HisLCb captured by
GST-HIP1h is shown in lane 4 in Figures 3(a) and 3(b), and as
expected, required bound LCb (compare lanes 3 and 4 in Figures 3(a) and 3(b)). We did not remove the
histidine tag on LCb used for purification because control experiments showed
that the tag did not interfere with the GST pulldowns (data not shown).
The binding of 6Hishub/6HisLCb was
significantly blocked when HIP1h K494 was replaced with glutamic acid (K494E
mutant, lane 5 in Figure 3(a)) or with alanine
(lane 5, Figure 3(b)).  In contrast, we saw no detectable impact when
R500, directly above K494 (see Figure 1 for location), was changed to glutamic
acid (see lane 6, Figure 3(a)) or alanine (data not shown).  Consistent with data in lanes 5 and 6 in
Figure 3(a), the K494E/R500E double mutant did not bind 6Hishub/6HisLCb (lane 7,
Figure 3(a)).  Next we evaluated a group of
basic residues close to the C-terminal end of S3 path (indicated by an arrow in
Figure 1).  Lanes 8 and 9 in Figure 3(a)
show that the R508E and K511E charge flip mutants bound 6Hishub/6HisLCb similar
to the wild type control (compare lane 4 with lanes 8-9 in Figure
3(a)).  We conclude from these data that
K494 located in the hydrophobic S3 path is required to bind CLC, but R500, R508,
and K511 around this path do not participate.
This suggests that the CLC binding site does not go beyond the
boundaries of S3 path that is defined by R500, R508, and K511.Two basic patches in HIP1 are critical for
binding clathrin light chain.  Western
blots were performed to analyze GST-pulldown experiments. Samples were run on
the same gel, but after transfer, the membrane was cut apart and blotted with
the appropriate antibody.  Bovine
clathrin heavy chain (aa1074-1675, Hub) with an N-terminal histidine-tag was
blotted with a commercial anti-His monoclonal antibody (Anti-His).  The neuronal form of clathrin light chain b
(LCb) had an N-terminal histidine-tag, but we used CON.1 to identify LCb.  The GST alone and GST-HIP1h constructs were
detected using a commercial monoclonal antibody against GST (Anti-GST).  Panel A: lanes 1 and 2: GST alone negative controls
show that there are no nonspecific interactions with 6Hishub alone or
6Hishub + 6HisLCb.  Lane 3: wild type
GST-HIP1h pulldown of 6Hishub alone (no bound LCb) indicates that the
interaction needs light chain.  Lane 4:
positive control, wild type GST-HIP1h with 6Hishub/6HisLCb.  Lanes 5–9: pulldowns of HIP1h single and double mutants
with 6Hishub/6HisLCb.  Panel B: lanes 1–3 are controls as
described in panel A.  Lane 4: positive
control, wild type GST-HIP1h with 6Hishub/6HisLCb.  Lane 5: GST-HIP1h K494A with
6Hishub/6HisLCb.  Lane 6: GST-HIP1h K474A
with 6Hishub/6HisLCb.
(a)(b)The assembled
HIP1 model (2NO2 joined to 2QA7) in Figure1 shows how the positive patch
centered on K474 is oriented relative to K494 (separated by ~50 Å).  Our data in Figure 3(b) show a dramatic drop in
the binding of 6Hishub/6HisLCb when K474 was mutated to alanine (see lanes 4
and 6 (K494A in lane 5 for comparison).
This result demonstrates for the first time that  K474 upstream the  DLL486R487KN
region is necessary for binding 6Hishub/6HisLCb and
suggests that the CLC binding site may be more extensive than previously
thought.  We predict that the two basic
patches in HIP1 we have defined here are also present in HIP12/R because K474
and K494 (HIP1 numbering) are both conserved in this protein.  Finally, we looked if K474, R487, and K494
were conserved in HIP1 from veterbrates Mus
musculus, Rattus norvegicus, Xenopus laevis, Drosophila melanogaster, and in sla2p (HIP1 homolog from Saccharomyces
cerevisiae) and ZK370.3 (HIP1 homolog from Caenorhabditis elegans).
This analysis showed that K474 was conserved in all the vertebrates but
was V in C. elegans, L in D. melanogaster, and M in sla2p.  R487 was conserved in all the vertebrates but
was E in both D. melanogaster and sla2p and T in C. elegans.  K494 was
conserved in all vertebrates and invertebrate HIP1 proteins, except sla2p (K474 (Q in S. cerevisiae), R487 (T), and K494 (D)).  The natural mutations in sla2p, HIP1 from D.
melanogaster, and ZK370.3 from C.
elegans could mean that the binding of HIP1 and HIP1 homologs to clathrin
light chain in these organisms is different.


## 2.1. Coiled-Coil Segment of HIP1 that Includes Determinants for Clathrin Light Chain Is Intrinsically Unstable

The binding of CLC to HIP1 requires L486 or R487 [7] (human
HIP1 numbering (accession number NP 005329), conserved in HIP12/1R).  We recently located L486 and R487 to a
flexible segment of HIP1 (position 2, Figure 1), in S3 path [8].  Here we used CD to probe the stability of
HIP1 468-530 (marked by the grey strip in Figure 1, between Y468 and R547), a
coiled-coil segment that has the S3 path.
At 4°C the CD profile was helical, indicated by the double minimum at
208 and 222 nm (Figure 2).  The ratio of
intensities at 222 and 208 nm can be used to distinguish coiled coils from
isolated helices (≥1 for coiled coils and <0.86 for
isolated helices [10, 11]).  The ratio
values in Figure 2 suggest that HIP1 468-530 shifts from a mixture of isolated
helices and coiled coils at low temperatures to isolated helices at 37°C.  We point out
that the 37°C profile is almost identical to the 83°C scan, suggesting that HIP1
468-530 contains a heat-stable element. We are presently doing NMR experiments to
map out the extent of the heat unstable portion of HIP1 468-530.Figure 1
Structural features of the HIP1 clathrin
light chain-binding site.  Two distantly
separated basic amino acids in the opened region of HIP1 mediate the binding of
clathrin light chain.  The assembled
model was generated using PDB files 2NO2 [8] and 2QA7 [9].  For the sake of clarity, we only show a
portion of the second HIP1 helix (dark grey) that stops before Y468. The yellow
bar marks the position of the solvent exposed hydrophobic S3 path [8].  The new data in Figure 3 indicate that K474
is a strong determinant for binding and imply that S3 path begins before the DLL486R487KN
region.  The N- and C-termini of the HIP1
crystal structure are labeled N and C.
The numbers 1–3 along the
yellow bar mark the position of amino acids that control the binding of
clathrin light chain (position 1: K474; position 2: L486 and R487 reported by
the McPherson group [7]; and position 3: K494).
K474 and K494 are ~50 Å apart.
R500, R508 or K511 (see arrow) do not participate in binding and
therefore define the boundary of the light chain-binding site.  The HIP1 468-530 subfragment
used in the CD studies in Figure 2 spans across an opened region of the HIP1
coiled coil in our 2NO2 and 2QA7 crystal structures.  The HIP1 model was created using PyMol
(http://www.pymol.org).Figure 2
HIP1 468-530 subfragment in the opened region is stable only at low
temperature.  Equilibrium far UV CD scans
were performed at4°C
(open triangle), 10°C (open diamond), 37°C (cross), and 83°C (open
circle).  The position of the 222 nm
signal did not change with temperature, but the 208 nm signal shifted as the
sample was heated.  The 222/208 nm ratios
were calculated from the raw CD data.
Because the signal below 200 nm was noisy, we did not attempt to get the
helix content from the CD data.


## 2.2. Basic Patches Centered on K474 and K494 in HIP1 Are Essential for Binding Clathrin Light Chain

We
investigated if a series of basic patches in the opened region could
participate in binding CLC.  K494 (see position
3, Figure1) is in S3 path (yellow bar, Figure 1) previously described [8] and
is followed by a cluster of basic residues (indicated by the arrow in Figure
1).  K474 (position 1) is located before
the DLL486R487KN region, close to the part of HIP1 that
mediates the binding of Huntingtin interacting protein 1 interactor (HIPPI)
[9].  To probe if those in S3 path contributed, we made 5 GST-HIP1h
(HIP1h is aa370-644) 370-644 mutants (K494A, K494E, R500E, R508E, and
K511E).  We performed GST pulldowns to
evaluate the binding of 6Hishub/6HisLCb (hub is central third of clathrin
(aa1074-1675) [12] and 6HisLCb is N-terminally histidine-tagged bovine clathrin
light chain b with the neuronal insert).
It is important to study 6HisLCb that is bound to 6Hishub to closely
mimic how HIP1 interacts with clathrin baskets in cells.  Every GST pulldown was done at least three
times, using freshly isolated proteins and charged GST beads each time.  Clathrin hub (N-terminally histidine tagged)
was detected by western blotting with a commercial histidine tag antibody.  LCb was blotted with CON.1 monoclonal
antibody and GST-HIP1h constructs were visualized with a commercial GST
antibody.  The anti-GST bands in Figures 3(a) and 3(b) showed that
the GST-HIP1h levels were balanced (loading control).  The negative controls in lanes 1–3 in Figure 3(a)
and lanes 1–3 in Figure 3(b)
show that the GST signals from each binding experiment were not random
interactions, but reflected true binding events.  The level of 6Hishub/6HisLCb captured by
GST-HIP1h is shown in lane 4 in Figures 3(a) and 3(b), and as
expected, required bound LCb (compare lanes 3 and 4 in Figures 3(a) and 3(b)). We did not remove the
histidine tag on LCb used for purification because control experiments showed
that the tag did not interfere with the GST pulldowns (data not shown).
The binding of 6Hishub/6HisLCb was
significantly blocked when HIP1h K494 was replaced with glutamic acid (K494E
mutant, lane 5 in Figure 3(a)) or with alanine
(lane 5, Figure 3(b)).  In contrast, we saw no detectable impact when
R500, directly above K494 (see Figure 1 for location), was changed to glutamic
acid (see lane 6, Figure 3(a)) or alanine (data not shown).  Consistent with data in lanes 5 and 6 in
Figure 3(a), the K494E/R500E double mutant did not bind 6Hishub/6HisLCb (lane 7,
Figure 3(a)).  Next we evaluated a group of
basic residues close to the C-terminal end of S3 path (indicated by an arrow in
Figure 1).  Lanes 8 and 9 in Figure 3(a)
show that the R508E and K511E charge flip mutants bound 6Hishub/6HisLCb similar
to the wild type control (compare lane 4 with lanes 8-9 in Figure
3(a)).  We conclude from these data that
K494 located in the hydrophobic S3 path is required to bind CLC, but R500, R508,
and K511 around this path do not participate.
This suggests that the CLC binding site does not go beyond the
boundaries of S3 path that is defined by R500, R508, and K511.Two basic patches in HIP1 are critical for
binding clathrin light chain.  Western
blots were performed to analyze GST-pulldown experiments. Samples were run on
the same gel, but after transfer, the membrane was cut apart and blotted with
the appropriate antibody.  Bovine
clathrin heavy chain (aa1074-1675, Hub) with an N-terminal histidine-tag was
blotted with a commercial anti-His monoclonal antibody (Anti-His).  The neuronal form of clathrin light chain b
(LCb) had an N-terminal histidine-tag, but we used CON.1 to identify LCb.  The GST alone and GST-HIP1h constructs were
detected using a commercial monoclonal antibody against GST (Anti-GST).  Panel A: lanes 1 and 2: GST alone negative controls
show that there are no nonspecific interactions with 6Hishub alone or
6Hishub + 6HisLCb.  Lane 3: wild type
GST-HIP1h pulldown of 6Hishub alone (no bound LCb) indicates that the
interaction needs light chain.  Lane 4:
positive control, wild type GST-HIP1h with 6Hishub/6HisLCb.  Lanes 5–9: pulldowns of HIP1h single and double mutants
with 6Hishub/6HisLCb.  Panel B: lanes 1–3 are controls as
described in panel A.  Lane 4: positive
control, wild type GST-HIP1h with 6Hishub/6HisLCb.  Lane 5: GST-HIP1h K494A with
6Hishub/6HisLCb.  Lane 6: GST-HIP1h K474A
with 6Hishub/6HisLCb.
(a)(b)The assembled
HIP1 model (2NO2 joined to 2QA7) in Figure1 shows how the positive patch
centered on K474 is oriented relative to K494 (separated by ~50 Å).  Our data in Figure 3(b) show a dramatic drop in
the binding of 6Hishub/6HisLCb when K474 was mutated to alanine (see lanes 4
and 6 (K494A in lane 5 for comparison).
This result demonstrates for the first time that  K474 upstream the  DLL486R487KN
region is necessary for binding 6Hishub/6HisLCb and
suggests that the CLC binding site may be more extensive than previously
thought.  We predict that the two basic
patches in HIP1 we have defined here are also present in HIP12/R because K474
and K494 (HIP1 numbering) are both conserved in this protein.  Finally, we looked if K474, R487, and K494
were conserved in HIP1 from veterbrates Mus
musculus, Rattus norvegicus, Xenopus laevis, Drosophila melanogaster, and in sla2p (HIP1 homolog from Saccharomyces
cerevisiae) and ZK370.3 (HIP1 homolog from Caenorhabditis elegans).
This analysis showed that K474 was conserved in all the vertebrates but
was V in C. elegans, L in D. melanogaster, and M in sla2p.  R487 was conserved in all the vertebrates but
was E in both D. melanogaster and sla2p and T in C. elegans.  K494 was
conserved in all vertebrates and invertebrate HIP1 proteins, except sla2p (K474 (Q in S. cerevisiae), R487 (T), and K494 (D)).  The natural mutations in sla2p, HIP1 from D.
melanogaster, and ZK370.3 from C.
elegans could mean that the binding of HIP1 and HIP1 homologs to clathrin
light chain in these organisms is different.


## 3. Materials and Methods

### 3.1. Materials

Triton X-100, Tween-20, beta-mercaptoethanol
(βME), TRIZMA base, and BIS-TRIS were from
Sigma-Aldrich (St. Louis, Mo, USA).
Sodium phosphate dibasic (Na2HPO4) was from EMD
Chemicals (Gibbstown, NJ, USA); Luria broth was from EMD Biosciences (Sparks, Md,
USA); tris(2-carboxyethyl)-phosphine was from Sigma-Aldrich. Pfu turbo was from Stratagene (La Jolla, Calif,
USA) and primers were from Integrated DNA Technologies (Coralville, Iowa, USA).  The Pierce Coomassie Plus Bradford reagent
kit was purchased from Fisher Scientific (Hanover Park, Ill, USA).  Chromatography resins, columns, and standards
were purchased from GE Healthcare (Piscataway, NJ, USA).  CON.1 antibody was bought from Covance
(Cumberland, Va, USA); restriction grade thrombin and the anti-His antibody
were obtained from Novagen (La Jolla, Calif, USA).  Coomassie G-250 stain and Immun-Star chemiluminescent kit were from Bio-Rad
Laboratories (Hercules, Calif, USA).


### 3.2. Construction of GST-HIP1h Mutants and 6HisLCb

The plasmid encoding the original N-terminal
GST tagged HIP1h 370-644 was a gift from the McPherson group.  The various GST-HIP1h mutants used in this
work were created using the QuikChange mutagenesis protocol (Stratagene). The
sequence was confirmed by DNA sequencing (IMBI, Indiana University) and then
transformed into Rosetta 2 (DE3) pLysS cells (Novagen).  Standard cloning was used to insert neuronal
LCb DNA inpET15b to generate
6HisLCb.  The recombinant 6HisLCb plasmid
was transformed into BL21 (DE3) pLysS cells for overexpression.


### 3.3. Protein Overexpression and Purification

The recombinant GST-HIP1h constructs were
grown at37°C in 1 L Luria broth (LB) to an O.D. 600 of 0.5–0.8 units.  The incubation temperature was dropped to 30°C cells and protein expression was induced with IPTG (100 μg/mL final concentration).  Cells were harvested after 3 hours at 30°C
and bacterial pellets were frozen at −80°C before use.  Bacterial pellets were resuspended in 50 mL of
1X PBS (10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.3), 140 mM NaCl, 2.7 mM KCl, supplemented with 0.25 mL of
1 M DTT, 0.25 mL of protease inhibitor cocktail (Sigma), and 2 mL
of PMSF (17.4 mg/mL in 2-propanol)).
After sonication, 2.5 mL of 20% (v/v) Triton X-100 was added and
the lysate was rotated at room temperature for ~30 minutes.  The crude bacterial lysate was spun at 12 000 g (4°C) for 10 minutes.  The supernatant was mixed with ~5 mL of
glutathione Sepharose 4B (Amersham) resin suspended in PBS.  The GST-HIP1h constructs were eluted from the
column with 50 mL of 3 mg/mL L-glutathione (sigma) in (PBS) at pH 8.0 and
dialyzed overnight against the same buffer.
For CD experiments the purification protocol was modified so that we can
cleave the GST tag in the GST column.
After bacterial lysate was added to GST beads, the mixture was rocked at
room temperature for 1-2 hours.  The beads were spun down at 500 g at 4°C for 5 minutes and the
supernatant was poured off.  The wet
beads were transferred to a column and washed slowly with 110 mL 1X PBS and
then 1 unit thrombin per mg of protein was diluted into 3.5 mL of 1X PBS and
added to the column.  After digestion,
the HIP1h constructs were further purified on Superdex 75 column (GE
Healthcare) equilibrated with 1X PBS (at room temperature).  Column fractions were pooled and dialyzed at
4°C against 10 mM potassium phosphate buffer at pH 7.9.The clathrin
hub construct was purified as previously described [13] and the 6HisLCb was
purified in a single step using the nickel affinity resin.  After the crude lysate was added to charged
Sepharose nickel resin and incubated at 4°C for 10 minutes, the beads were
gently spun down and washed with 25 mL of 10 mM Na2HPO4,
10 mM imidazole, 0.5 M NaCl, pH 7.4 buffer (buffer A).  The beads were then washed with 30 mL of buffer
A that contained 0.5 M imidazole.  The
6HisLCb was eluted off the column with buffer A that contained 205 mM
imidazole.  EDTA was added to the sample
(1 mL 0.5 M EDTA per 10 mL of protein) and dialyzed against 10 mM tris, pH 7.9
overnight at 4°C (dialysis buffer includes BME, and a cocktail of protease
inhibitor).


### 3.4. CD Measurements

Purified HIP1 468-530 was diluted with 10 mM
potassium phosphate buffer at pH 7.9 to 0.5 mg/mL for CD measurements at
different temperatures.  CD data was
collected using a Jasco J-175 circular dichroism spectropolarimeter with
thermally controlled sample cells.  The
first CD scan was taken at4°C and then the temperature was changed to the
indicated temperatures in Figure 2.  The
sample was allowed to sit at each indicated temperature for several minutes
before taking the CD scan.


### 3.5. GST Pull Down Assays

Glutathione Sepharose 4B resin (GE
Healthcare 17-0756-01) was washed three times with 1 mL of PBS.  Protein concentrations were determined by
Bradford assays (Pierce 23236).  Equal
molar amounts of GST and GST-Hip1h proteins (1μM) and 1 mL of PBS were added to resin and
incubated on a rotating platform at 4°C for 1 hour.  Unbound proteins were removed by washing
three times with binding buffer (50 mM Tris, 200 mM KCl, 1 mM EDTA, 1% Triton X-100,
50 mM imidazole, 0.5 mg/mL ovalbumin, pH 8.0).
6HisLCb and Hub alone were combined in a 3:1 ratio and allowed to
incubate at 4°C for at least 30 minutes.
25 μL of GST or GST-Hip1h bound beads, 0.4 nmol
of Hub Alone or 1.9 nmol of Hub 6HisLCb complex, 375 μL of room temperature binding buffer were
added to illustra MicroSpin columns (GE Healthcare 27-3565-01) and incubated on
rotator at 4°C for 1 hour.  Beads were
washed six times with 0.5 mL of binding buffer + 16 mM imidazole and supernatant
removed by centrifugation.  After final
wash, 55 μL of 2x SDS gel loading buffer was added to
beads. Spin columns were closed placed in 1.7 mL Eppendorf tubes, heated for 10
minutes at 80–90°C, and spun
down to collect samples.


### 3.6. Western Blots

The bound proteins were resolved by SDS-PAGE
and analyzed by standard western blotting.
After transfer the nitrocellulose membrane was stained with Ponceau
stain and cut to separate proteins for blotting.  Hub was detected with anti-His monoclonal antibody
(Novagen 70796-3), GST and GST-Hip1h were detected with anti-GST monoclonal
antibody (Covance MMS-112P), and 6HisLCb was detected with clathrin light chain monoclonal
antibody (CON.1) (Covance MMS-423P).
Binding was detected with Immun-Star chemiluminescent protein detection system
(BioRad 170-5010).


## 3.1. Materials

Triton X-100, Tween-20, beta-mercaptoethanol
(βME), TRIZMA base, and BIS-TRIS were from
Sigma-Aldrich (St. Louis, Mo, USA).
Sodium phosphate dibasic (Na2HPO4) was from EMD
Chemicals (Gibbstown, NJ, USA); Luria broth was from EMD Biosciences (Sparks, Md,
USA); tris(2-carboxyethyl)-phosphine was from Sigma-Aldrich. Pfu turbo was from Stratagene (La Jolla, Calif,
USA) and primers were from Integrated DNA Technologies (Coralville, Iowa, USA).  The Pierce Coomassie Plus Bradford reagent
kit was purchased from Fisher Scientific (Hanover Park, Ill, USA).  Chromatography resins, columns, and standards
were purchased from GE Healthcare (Piscataway, NJ, USA).  CON.1 antibody was bought from Covance
(Cumberland, Va, USA); restriction grade thrombin and the anti-His antibody
were obtained from Novagen (La Jolla, Calif, USA).  Coomassie G-250 stain and Immun-Star chemiluminescent kit were from Bio-Rad
Laboratories (Hercules, Calif, USA).


## 3.2. Construction of GST-HIP1h Mutants and 6HisLCb

The plasmid encoding the original N-terminal
GST tagged HIP1h 370-644 was a gift from the McPherson group.  The various GST-HIP1h mutants used in this
work were created using the QuikChange mutagenesis protocol (Stratagene). The
sequence was confirmed by DNA sequencing (IMBI, Indiana University) and then
transformed into Rosetta 2 (DE3) pLysS cells (Novagen).  Standard cloning was used to insert neuronal
LCb DNA inpET15b to generate
6HisLCb.  The recombinant 6HisLCb plasmid
was transformed into BL21 (DE3) pLysS cells for overexpression.


## 3.3. Protein Overexpression and Purification

The recombinant GST-HIP1h constructs were
grown at37°C in 1 L Luria broth (LB) to an O.D. 600 of 0.5–0.8 units.  The incubation temperature was dropped to 30°C cells and protein expression was induced with IPTG (100 μg/mL final concentration).  Cells were harvested after 3 hours at 30°C
and bacterial pellets were frozen at −80°C before use.  Bacterial pellets were resuspended in 50 mL of
1X PBS (10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.3), 140 mM NaCl, 2.7 mM KCl, supplemented with 0.25 mL of
1 M DTT, 0.25 mL of protease inhibitor cocktail (Sigma), and 2 mL
of PMSF (17.4 mg/mL in 2-propanol)).
After sonication, 2.5 mL of 20% (v/v) Triton X-100 was added and
the lysate was rotated at room temperature for ~30 minutes.  The crude bacterial lysate was spun at 12 000 g (4°C) for 10 minutes.  The supernatant was mixed with ~5 mL of
glutathione Sepharose 4B (Amersham) resin suspended in PBS.  The GST-HIP1h constructs were eluted from the
column with 50 mL of 3 mg/mL L-glutathione (sigma) in (PBS) at pH 8.0 and
dialyzed overnight against the same buffer.
For CD experiments the purification protocol was modified so that we can
cleave the GST tag in the GST column.
After bacterial lysate was added to GST beads, the mixture was rocked at
room temperature for 1-2 hours.  The beads were spun down at 500 g at 4°C for 5 minutes and the
supernatant was poured off.  The wet
beads were transferred to a column and washed slowly with 110 mL 1X PBS and
then 1 unit thrombin per mg of protein was diluted into 3.5 mL of 1X PBS and
added to the column.  After digestion,
the HIP1h constructs were further purified on Superdex 75 column (GE
Healthcare) equilibrated with 1X PBS (at room temperature).  Column fractions were pooled and dialyzed at
4°C against 10 mM potassium phosphate buffer at pH 7.9.The clathrin
hub construct was purified as previously described [13] and the 6HisLCb was
purified in a single step using the nickel affinity resin.  After the crude lysate was added to charged
Sepharose nickel resin and incubated at 4°C for 10 minutes, the beads were
gently spun down and washed with 25 mL of 10 mM Na2HPO4,
10 mM imidazole, 0.5 M NaCl, pH 7.4 buffer (buffer A).  The beads were then washed with 30 mL of buffer
A that contained 0.5 M imidazole.  The
6HisLCb was eluted off the column with buffer A that contained 205 mM
imidazole.  EDTA was added to the sample
(1 mL 0.5 M EDTA per 10 mL of protein) and dialyzed against 10 mM tris, pH 7.9
overnight at 4°C (dialysis buffer includes BME, and a cocktail of protease
inhibitor).


## 3.4. CD Measurements

Purified HIP1 468-530 was diluted with 10 mM
potassium phosphate buffer at pH 7.9 to 0.5 mg/mL for CD measurements at
different temperatures.  CD data was
collected using a Jasco J-175 circular dichroism spectropolarimeter with
thermally controlled sample cells.  The
first CD scan was taken at4°C and then the temperature was changed to the
indicated temperatures in Figure 2.  The
sample was allowed to sit at each indicated temperature for several minutes
before taking the CD scan.


## 3.5. GST Pull Down Assays

Glutathione Sepharose 4B resin (GE
Healthcare 17-0756-01) was washed three times with 1 mL of PBS.  Protein concentrations were determined by
Bradford assays (Pierce 23236).  Equal
molar amounts of GST and GST-Hip1h proteins (1μM) and 1 mL of PBS were added to resin and
incubated on a rotating platform at 4°C for 1 hour.  Unbound proteins were removed by washing
three times with binding buffer (50 mM Tris, 200 mM KCl, 1 mM EDTA, 1% Triton X-100,
50 mM imidazole, 0.5 mg/mL ovalbumin, pH 8.0).
6HisLCb and Hub alone were combined in a 3:1 ratio and allowed to
incubate at 4°C for at least 30 minutes.
25 μL of GST or GST-Hip1h bound beads, 0.4 nmol
of Hub Alone or 1.9 nmol of Hub 6HisLCb complex, 375 μL of room temperature binding buffer were
added to illustra MicroSpin columns (GE Healthcare 27-3565-01) and incubated on
rotator at 4°C for 1 hour.  Beads were
washed six times with 0.5 mL of binding buffer + 16 mM imidazole and supernatant
removed by centrifugation.  After final
wash, 55 μL of 2x SDS gel loading buffer was added to
beads. Spin columns were closed placed in 1.7 mL Eppendorf tubes, heated for 10
minutes at 80–90°C, and spun
down to collect samples.


## 3.6. Western Blots

The bound proteins were resolved by SDS-PAGE
and analyzed by standard western blotting.
After transfer the nitrocellulose membrane was stained with Ponceau
stain and cut to separate proteins for blotting.  Hub was detected with anti-His monoclonal antibody
(Novagen 70796-3), GST and GST-Hip1h were detected with anti-GST monoclonal
antibody (Covance MMS-112P), and 6HisLCb was detected with clathrin light chain monoclonal
antibody (CON.1) (Covance MMS-423P).
Binding was detected with Immun-Star chemiluminescent protein detection system
(BioRad 170-5010).



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*Source: 256124-2009-04-07.xml*