(Received for publication, June 13, 1995; and in revised form, July 10, 1995)
From the
The oxygen-avid, perienteric hemoglobin of Ascaris is a homooctamer. Each subunit contains two tandem globin domains that are highly homologous with the exception of a charged COOH-terminal extension. In solution, recombinant domain one (D1) exists as a monomer, whereas recombinant domain two with the COOH-terminal tail (D2) is primarily an octamer. To examine the role of the COOH-terminal extension in Ascaris hemoglobin multimer formation, we attached the tail to the monomeric, heme-containing proteins, myoglobin and D1; neither construct was capable of multimer formation. Additionally, we removed the tail from both full-length Ascaris hemoglobin and D2. This substantially decreased, but did not eliminate, multimerization. We further characterized subunit interactions by disrupting full-length hemoglobin multimers with the chaotropic salt, NaSCN, which yielded intermediate oligomers. In solution, D2 demonstrated a greater propensity to dissociate than full-length hemoglobin, indicating that D1 contributes to octamer stability. D1 formed a weak dimer in its crystal; thus, we analyzed interactions along the subunit interface. Hydrogen bonds as well as hydrophobic and electrostatic forces appeared to contribute to dimer formation. Amino acid substitutions along this interface in D2 are predicted to enhance subunit interactions for that domain. Our studies reveal that the COOH-terminal tail is necessary, but not sufficient, for efficient octamer formation. Other regions, possibly within the E- and F-helices and AB loops of both domains, appear to be important for Ascaris hemoglobin octamer formation.
The perienteric fluid of the parasitic nematode Ascaris contains an abundant hemoglobin with exceptional oxygen avidity(1) . This globin is an octamer composed of identical 43-kDa polypeptides(2, 3) . Each subunit consists of two tandem globin domains. A highly charged tail is found at the COOH terminus of the second domain (Fig. 1A)(4, 5) . The crystal structure of the first globin domain has been solved, revealing striking similarities to the tertiary structures of globins from diverse species(6) . The heme pocket contains a network of hydrogen bonds, including one contributed by a critical tyrosine, which locks the liganded oxygen in place. Mutagenesis studies have provided further support for this(7, 8) .
Figure 1:
Ascaris hemoglobin
constructs. A, the primary structures of D1 and D2 are aligned
with the charged COOH-terminal tail highlighted in bold(4, 9) . D1 contains amino acids
1-149 (M
17,900). D2 contains
151-320 (M
20,600). B, a
two-step PCR strategy was utilized in order to engineer D1 with the
tail (D1+t). In the first step, complementary oligonucleotide
primers (Primers b and b`) containing regions complementary to both the
extreme 3` end of D1 and the extreme 5` end of the tail were used in
separate reactions with primers complementary to the 5` end of D1
(Primer a in Reaction I) or the 3` end of the COOH-terminal tail
(Primer c in Reaction II). The products, containing a region of shared
overlap, were utilized as template in the next step with only the
extreme 5` and 3` end-specific primers (Primers a and c) to generate
full-length D1+t.
Although the
molecular basis of the oxygen avidity of Ascaris hemoglobin
has now been described in some detail, structural motifs responsible
for the formation of this remarkable multimer have not yet been
elucidated. However, molecular cloning and expression of the separated
domains have provided interesting clues (9) . Domain one (D1),
which does not have the COOH-terminal extension, exists as a monomer in
solution. In contrast, domain two (D2), which contains the tail, is
capable of polymerizing to an octamer. Excluding the tail, the primary
structures of D1 and D2 are highly homologous with 63% amino acid
identity, implicating the tail as a potentially important component in
multimerization(4, 5) . The 23-residue COOH-terminal
extension is highly polar, possessing 22 charged residues, including
four His-Lys-Glu-Glu (HKEE) direct repeats. It has been suggested that
this tail may serve as a polar zipper, forming a -barrel comprised
of eight antiparallel
-strands stabilized by interstrand salt
bridges between side chains(8, 10) . Electron
microscopy and cross-linking studies have indicated that the octamer
conforms to a two-layered arrangement of subunits, perhaps with one
type of interaction of subunits within a layer and another type between
the two layers(3) . In this study, we have characterized the
interaction of subunits and have assessed the contribution of the
highly charged tail in Ascaris hemoglobin octamer formation.
D2 lacking the COOH-terminal tail (D2-t) was constructed using the D2 expression plasmid (9) as a template in PCR. The D2-t forward primer was the same used for amplification of D2(9) . The D2-t reverse primer was the one used above for the formation of AH-t.
Addition of the COOH-terminal tail to D1 in order to create D1+t was performed utilizing a two-step PCR-based strategy modified from Morrison and Desrosiers (11) (Fig. 1B). In the first PCR step, Reaction I used the D1 expression plasmid as template(9) . Primer a, the forward primer, contained an NcoI restriction site and was the same used for amplification of D1 as described previously(9) . Primer b, the reverse primer in this reaction (5` ttctttatgctcgtggtgttctcgtccatgcttgttgatttc 3`), contained 21 nucleotides complementary to the extreme 3` end of the coding sequence for D1, plus 21 nucleotides complementary to the extreme 5` coding sequence for the tail. In Reaction II, the D2 expression plasmid was used as template. Primer b`, the complement to Primer b, was used as the forward primer. Primer c, the reverse primer, contained an NcoI restriction site and was same utilized for the amplification of D2 as described previously(9) . Reactions I and II resulted in products with a region of shared overlap with one another. In order to generate full-length D1+t, a 10-fold dilution of both products was used as template in Reaction III with only the extreme 5` and 3` end-specific oligonucleotides, Primers a and c. In the first round of amplification, the single-stranded templates hybridized in the region of shared overlap and self-primed the extension reaction, thus creating the desired full-length double-stranded insert. In all subsequent rounds, the 5`- and 3`-specific primers amplified this product.
PCR was performed in a
TC480 thermal cycler (Perkin-Elmer). Final reaction conditions in all
PCR were 1.25 units of AmpliTaq (Perkin-Elmer), 0.1 µM oligonucleotide primers, 20 mM Tris-HCl (pH 8.2), 10
mM KCl, 6 mM
(NH)
SO
, 2 mM MgCl
, 0.1% Triton X-100, 10 µg/ml nuclease-free
bovine serum albumin, 0.2 mM deoxynucleotides, and the
relevant template in a final volume of 0.05 ml. Reactions were cycled
30 times at 95 °C for 1 min, 50 for 1 min, and 75 for 1 min. After
ethanol precipitation, PCR products were digested at 37 for 16 h with
10 units of NcoI (Boehringer Mannheim). The DNA was then
purified by adsorption to a glass slurry using the Elu-Quik DNA
purification kit (Schleicher & Schuell) and cloned into NcoI-digested, phosphatase-treated Studier expression vector
pET-8C. Phosphatase reactions were carried out at for 1 h using 1 unit
of calf intestinal phosphatase
Expression constructs were transformed into Escherichia coli strain XL1-Blue for plasmid screening. Positive clones were selected on LB-ampicillin plates and were screened for forward orientation by PCR analysis. Positives were verified for the expected mutations by plasmid DNA sequencing with the Sequenase kit (U. S. Biochemical Corp.), and the products were resolved on 6% polyacrylamide gels (National Diagnostics).
Further purification of hemoglobins for NaSCN dissociation experiments was accomplished by dilution of the samples 1:40 in 20 mM Tris (pH 7.5) and fractionation using Mono Q anion exchange chromatography. A Pharmacia Mono Q HR 5/5 column was equilibrated with 25 mM Tris (pH 7.5), 50 mM NaCl for 15 min, and samples were eluted with a linear gradient from 50 mM to 1000 mM NaCl for 30 min at a flow rate of 1 ml/min. Samples were then concentrated using Centriprep 10 concentrators.
Figure 2: Quaternary structure of myoglobin with the tail. Mgb+t (monomer mass, 21 kDa) was injected onto a Superose 12 gel filtration column as described under ``Experimental Procedures.'' Elution times (in minutes) were compared with those of molecular weight standards. The 17.5-kDa standard corresponds to myoglobin without a tail. Myoglobin elution from the column was monitored by absorbance at 410 nm.
Figure 3: Quaternary structure of Ascaris hemoglobin constructs. Samples were injected onto a Superose 12 gel filtration column as described under ``Experimental Procedures.'' Elution times (in minutes) were compared with those of molecular weight standards. Hemoglobin elution from the column was monitored by absorbance at 410 nm. A, comparison of D1 (bold line, monomer mass 18 kDa) with D1+t (thin line, monomer mass 21 kDa). B, comparison of AH (bold line; monomer mass, 39 kDa) with AH-t (thin line; monomer mass, 36 kDa); C, comparison of D2 (bold line; monomer mass, 21 kDa) with D2-t (thin line; monomer mass, 18 kDa). Time is shown in minutes on the x axis.
Figure 4: Disruption of octameric Ascaris hemoglobin. Ascaris hemoglobin was incubated in the presence of varying concentrations of NaSCN for 20 min as described under ``Experimental Procedures.'' Samples were then injected onto a Superose 12 gel filtration column. Elution times (in minutes) were compared with those of molecular weight standards. Hemoglobin elution from the column was monitored by absorbance at 410 nm. A, control with no added NaSCN; B, 1 M NaSCN; C, 1.5 M NaSCN; D, 2 M NaSCN. The dashed line corresponds to the position of eluted octamer. Expected positions are indicated for tetramer (t), dimer (d), and monomer (m).
In the next part of this experiment, purified full-length hemoglobin was incubated in the presence of 2 M NaSCN for increasing amounts of time to induce further dissociation of multimers. Quaternary structure was again assessed by gel filtration chromatography (Fig. 5). At 20 min, as in Fig. 4D, substantial dissociation into dimer and tetramer was observed. By 60 min, there was additional loss of octamer and tetramer populations with a striking increase in dimer. Upon incubation for 90 min, more substantial dissociation into monomer was observed. Similar results were obtained upon incubation for 135 min (data not shown). After 180 min, no peaks were observed, due to subunit denaturation with subsequent loss of the heme moiety.
Figure 5: Dissociation of Ascaris hemoglobin multimers. Ascaris hemoglobin was incubated for varying times in 2 M NaSCN (final concentration) as described under ``Experimental Procedures.'' Samples were then injected onto a Superose 12 gel filtration column. Elution times (in minutes) were compared with those of molecular weight standards. Hemoglobin elution from the column was monitored by absorbance at 410 nm. A, control incubated for 20 min with no added NaSCN; B, 20-min incubation; C, 60-min incubation; D, 90-min incubation; E, 180-min incubation. Expected positions are indicated for tetramer (t), dimer (d), and monomer (m).
Figure 6: Stereo diagram of the D1 dimer interface. Helix backbone atoms of each D1 subunit are shown with the AB-loop and the E- and F-helices highlighted in bold.
Recently, the crystal structure of Ascaris hemoglobin domain one was reported(6) . However, because
D1 alone does not form multimers in solution(9) , this
structure has not defined regions of the full-length molecule involved
in octamer formation. The two tandem globin folds are highly homologous
with the exception of a highly charged, 23 residue COOH-terminal
extension on D2, which contains 22 ionizable residues, including four
direct HKEE repeats(4, 5) . Perutz and colleagues (5) have suggested that this tail may be capable of forming an
eight-stranded -barrel. Thus, we targeted the Ascaris hemoglobin tail as a potentially important structure required for
octamer formation.
We first tested the ability of the tail to
directly promote multimerization of a structurally related but
monomeric protein. Myoglobin served as an appropriate candidate, due to
its homologous compact, highly -helical
structure(6, 12) . The engineered myoglobin,
Mgb+t, was incapable of forming multimers. This raised the
possibility that the myoglobin either contained regions that interfered
with multimerization or lacked certain motifs necessary for multimer
formation. In order to address this, we constructed a mutant of a
completely unrelated molecule, glutathione S-transferase,
which upon addition of the charged tail was also incapable of forming
multimers (data not shown). Taken together, these data suggest that
interaction of other regions in the Ascaris hemoglobin
molecule are essential for multimerization. Indeed, construction and
analysis of D1+t demonstrated that the tail alone is unable to
promote multimer formation of the monomeric, Ascaris hemoglobin domain. In contrast, full-length hemoglobin without a
tail, as well as D2 without a tail, were still capable of forming the
full complement of multimers, albeit in a poorly controlled fashion.
The most abundantly observed multimer in these tail-less hemoglobins
was a dimer, suggesting that the regions responsible for dimer
formation lie within the globin fold of D2. Based on these data, we
propose that the ability to multimerize is intrinsic to the globin
region of D2 and that the COOH-terminal tail serves to stabilize the Ascaris hemoglobin octamer.
Previously, Darawshe and Daniel (3) proposed that Ascaris hemoglobin adopts a
two-layered arrangement with four monomers in each layer. Using
cross-linking analysis, the distribution of cross-linked species
corresponded to theoretical values predicted from a two-layered
arrangement with one type of interaction within a layer and another
type between layers. Based on these results, the investigators
suggested that a disruptive agent could possibly discriminate between
these interactions, yielding tetramer and dimer intermediates.
Additionally, the polar zipper model of Perutz and co-workers (5, 10) aligns four monomers on either side of the
-barrel, consistent with the arrangement proposed by Darawshe and
Daniel(3) . A previous investigation using pH extremes showed
dissociation of Ascaris hemoglobin directly into dimers and
monomers(3) . In our studies, a chaotropic salt, NaSCN, served
as a more selective reagent for disruption of the octamer. If only one
type of interaction was responsible for octamer formation, then the
multimer should dissociate directly into monomers. However, gel
filtration analysis yielded elution times corresponding to dimer as
well as tetramer, indicating that several types of interactions exist
within the octamer. In our NaSCN experiments, it is possible that some
subunit reassociation occurred during gel filtration analysis.
Regardless, the detection of intermediate species indicates the
existence of multiple types of interactions between subunits. These
data, combined with our analysis of the tail-less mutants, suggest that
interactions between the globin folds, in addition to those between the
charged tails, are important for octamer assembly.
Although D2 is
certainly capable of multimerization, its ability to form an octamer is
reduced in comparison to the full-length molecule, demonstrated by the
presence of detectable intermediates. Furthermore, upon dilution, D2
readily dissociated, whereas the full-length molecule remained
octameric (data not shown). These results suggest that the first domain
in native Ascaris hemoglobin may contribute additional
interactions required for the formation of a stable octamer, despite
the inability of separated D1 with the tail to multimerize. The fact
that D1 forms a weak dimer in the crystal provides further support for
this hypothesis. Distinct from vertebrate hemoglobin tetramer
interactions(12) , the D1 interface is formed by the E- and F-
helices along with the AB loop. Examination of surface residues at the
D1 dimer interface revealed numerous favorable hydrophobic and
electrostatic interactions. This dimer may represent actual molecular
interactions that occur between D1 subunits within native Ascaris hemoglobin in solution. Furthermore, an analogous interface may
serve as a major site of D2 subunit interactions. Interestingly,
Val and Ala
in D1 are replaced with leucine
and methionine, respectively, in D2. Both of these substitutions result
in side chains with greater hydrophobic surface areas than D1, which
could serve as a strong driving force promoting multimer formation.
In this study, mutagenesis experiments demonstrated that the COOH-terminal tail is required for stable octamer formation, but is not sufficient to promote multimerization alone. Dissociation experiments and analysis of the D1 crystalline dimer provided further insight into subunit interactions. Unquestionably, solution of the crystal structure of full-length Ascaris hemoglobin or D2 will provide the most direct approach for elucidating the structural basis of octamer formation. This will allow direct visualization of quaternary structure, includ-ing relevant contacts between globin domains, as well as interactions between the charged COOH-terminal tails.