PROTEINS Structure, Function, and Genetics 17:62-74 (1993)

A Homology Model of Human Interferon a-2 Nicholas J. Murgolo,' William T. Windsor,' Alan Hruza,' Paul Reichert,' Anthony Tsarbopoulos,' Samuel Baldwin,' Eric Huang,' Birendra Pramanik,' Steven Ealick? and Paul P. Trotta' 'BiotechmlogylBiochemistry and PACRD, Schering-Plough Research Institute, Kenilworth, New Jersey 07033; and 2Department of Biochemistry and Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

ABSTRACT An atomic coordinate five a-helix three-dimensional model is presented for human interferon 01-2 (HuIFNa2). The HuIFNa2 structure was constructed from mu- rine interferon p (MuIFNp) by homology mod- eling using the STEREO and IMPACT pro- grams. The HuIFNor2 model is consistent with its known biochemical and biophysical proper- ties including epitope mapping. Lysine residues predicted to be buried in the model were pri- marily unreactive with succinimidyl-7-amino-4- methylcoumarin-3-acetic acid (AMCA-NHS), a lysine modification agent, as shown by mass spectrometric analysis of tryptic digests. N-ter- minal sequence analysis of polypeptides gen- erated by limited digestion of HuIFNor2 with endoproteinase Lys-C demonstrated rapid cleavage at K31, which is consistent with the presence of this residue in a loop in the pro- posed H u I F N d model. Based on this model structure potential receptor binding sites are identified. o 1993 wiley-Liss, hc.

Key words: a interferon, p interferon, homol- ogy modeling

INTRODUCTION Human interferon a-2 (HuIFNa2) is a highly

pleiotropic cytokine with broad-spectrum antiviral, antiproliferative, and immunomodulatory proper- ties (reviewed in references 1-3). In particular, HuIFNaZ inhibits replication of a variety of RNA- and DNA-containing viruses, inhibits the growth of malignant cells, affects the expression of a variety of oncogenes, and activates natural killer cells. All of these properties are expressed through binding to specific, high-affinity cell surface receptors, which are widely distributed on various cell types.4 It is consistent with the biology of HuIFNa2 that Intron A is effective against a variety of cancer and viral indications in

Despite a wealth of data on its biological effects, the three-dimensional structure of HuIFNa2 has yet to be determined. The lack of a current three-dimen- sional model a t atomic resolution for HuIFNa2 con- sistent with its known properties prompted us to construct a homology model based on the recently determined three-dimensional structure of murine 0 1993 WILEY-LJSS, INC.

interferon p (MuIFNp).'p6 The identities in amino acid sequence between HuIFNa2 and MuIFNp, be- tween HuIFNa2 and HuIFNp, and between HuIFNp and MuIFNp are 29,33, and 49%, respec- tively (see Table I).' This high degree of sequence identity across species and interferon classes sug- gests that the proteins have a similar In ad- dition to significant sequence identity, IFNa and IFNp also share the same cell-surface receptor,10J1 and induce translation of similar factors.12

Secondary structural prediction algorithms are consistent with an a-helical content of 50% for HuIFNa2.13 Physical studies including circular di- chroism and Raman spectroscopy suggest HuIFNa2 contains 45-70% and 49% a-helix, re~pectively.'~.~' These percentages are comparable to the 49% a-helix content seen in the crystal structure of M U I F N ~ . ~ ~ ~ IFNa and IFNp have similar structural properties as well as a high cross-species amino acid sequence identity, permitting homology modeling of HuIFNaZ based on the known structure of MuIFNp. This model is consistent with the known biochemi- cal and biophysical properties of HuIFNa2, includ- ing the chemical modification and proteolytic cleav- age studies described here.

METHODS Molecular Modeling of MuIFNP

The crystal structure of MuIFNp has been solved to 2.6 A resol~tion.',~ The MuIFNP structure con- sists of 5 helices [residues 8-25 (A), 53-68 (B), 79-90 ( C ) , 114-131 (D), and 139-154 (E)]. Helices A, B, and D are oriented parallel to each other while helices C and E are antiparallel to A, B, and D. There is also a high degree of flexibility in the CD loop (89-111). Since the coordinates for MuIFNp

Abbreviations: HuIFNa2, human interferon a-% MuIFNp, murine interferon p; AMCA-NHS, succinimidyl-7-amino-4- methylcoumarin-3-acetic acid; "FA, trifluoroacetic acid; one letter codes are used to designate amino acids.

Received October 15, 1992; revision accepted March 23, 1993. ~~~

Address reprint requests to Dr. Nicholas J. Murgolo, Scher- ing-Plough Research Institute, 2015 Galloping Hill Road, Ke- nilworth, NJ 07033.

HOMOLOGY MODEL OF HuIFNa2 63

had not been disclosed at the onset of this study the published Ca backbone stereo diagram was used to generate a model ~ t ruc ture .~ Through use of the STEREO program publicly distributed with the Brookhaven Protein Database," a Ca backbone structure of MuIFNp was generated. The structure was extended to include backbone atoms and typical sidechain geometries with the Biopolymer Module of the Sybyl Program (Tripos Associates, St. Louis, MO).

The resulting structure for MuIFNp resembled the stereodiagram of the Ca atoms presented in the report of the crystal structure. A few structural in- accuracies were noted in this model structure prior to molecular mechanics minimization. For example, a large number of buried hydrophilic residues were noted in helix A (E10, R15, K16, E19, E22) that did not appear to participate in any H-bonds or ion pairs. Additionally, E86 in helix C, which was bur- ied, had no apparent partner. We rationalized that the unpaired acidic residue E86 could form an ion pair with R15 or K16 if helix A were rotated. The rotation included a two residue shift toward the C-terminus of helix A. This rotation resulted in the favorable formation of a K16-E86 ion pair, an H-bond between residues El0 and Q7, and permit- ted side chain exposure of hydrophilic residues R11, R15, E19, and E22 of helix A. Solvent exposure of the hydrophilic side of the adjusted helix A was con- sistent with expectations from helical wheel dia- grams (Fig. l) and with the recently reported hydro- philic residue exposures.6

Recently we obtained the Ca coordinates depos- ited at Brookhaven for the MuIFNP structure (pre- liminary entry PlIFA).' An orientation difference was noted between the MuIFNp A helix of the model structure and entry PlIFA such that the deposited coordinates resembled the earlier STEREO-gener- ated model prior to helix A adjustment, having a Ca rms of 1 A. Extension of the PlIFA crystal structure to include backbone atoms and typical side chain geometries showed helix A again contained buried ions. Further, we repeated the HuIFNa2 modeling with the PlIFA coordinates. This homology model of HuIFNa2 built from the PlIFA MuIFNP coordi- nates had similar structural inaccuracies involving buried hydrophilic residues, having a Ca rms of 1 A compared with the original model built from unro- tated STEREO-generated MuIFNP coordinates. Given the moderate 2.6 A resolution of the MuIFNp crystal structure: it is possible that the orientation of the A helix presented in the PlIFA entry may be incorrect due to poor definition in the crystal struc- ture.

The model for MuIFNp containing an adjusted he- lix A was refined by 500 cycles of constrained min- imization followed by 500 cycles of free molecular mechanics minimization using the IMPACT pro- gram." The AMBER all-atom force field was em-

Fig. 1. Helical wheel diagrams of HulFNaP (upper), MulFNP (lower) Hydrophobic residues are circled; charged residues are underlined if they participate in ion pairs or hydrogen bonds and boxed if they do not. The buried face of each helix is indicated by a dashed line. The orientation of each helix from N- to C-terminus is indicated with an arrow. For both models there is a high con- centration of hydrophobic residues in the buried core and all bur- ied charge residues participate in interactions. The amphipathic nature of helix A in the MulFNP model suggests the orientation presented in the current model is correct.

ployed with a distance-dependent dielectric constant in the electrostatic term. The constrained minimi- zation included i HN to i+3 CO and i HN to i + 4 CO NOE-type distance constraints of 2.71 2 0.25 A and 1.86 * 0.25 A, respectively, for helical residues. Tor- sional constraints were also employed in the con- strained refinement for helical residues by requiring phi values of 302" and psi values of 313". Interior helical residues were allowed a range of 30" and the endcaping N- and C-terminal residues were allowed a less stringent range of 60" for both phi and psi torsional constraints. The final refined model result- ing from the helix A rotation and 2 amino acid shift is consistent with known structural features of MuIFNp (see below). Molecular calculations were performed on either a Convex C220 minisupercom- puter or a Silicon Graphics 4D/240 computer. Molec- ular graphics inspection of models was performed on

TA

BL

E I

. Seq

uenc

e Alignment

of Human I

FN

a2, I

FNP,

and Murine

IFN

P*

>I

I

<-

--

--

HE

LIX

A -

- - -

-

1 10

20

30

I

I I

I Hu

man

IFNa

2 C

DL

PQ

TH

SL

GS

RR

TL

ML

LA

QM

RR

IS

LF

SC

LK

DR

HD

Huma

n IF

NP

MS

YN

LL

GF

LQ

RS

SN

FQ

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NG

RL

EY

CL

KD

RM

N

Muri

ne I

FNP

IN

YK

QL

QL

QE

RT

NI

RK

C'

QE

LL

EQ

LN

GK

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60

70

I I

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FG

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VL

HE

MI

QQ

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Huma

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FD

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IF

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- >

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80

90

100

I I

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2 S

SA

AW

DE

TL

LD

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YQ

QL

ND

LE

AC

VI

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Huma

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SS

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EE

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ED

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Muri

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VR

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FL

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90

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--

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- -

- -

>I

I

<-

--

110

120

130

140

I I

I I

Human IFNU2

PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRA

Human IFNP

TRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRV

Murine IFNP

TWEMSSTALHLKSYYWRVQRYLKLMKYNSYAWMVVRA

II

I

ll

1

I I

I I

ll

I

I

I I

II

I

II

I

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l

I I

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110

120

130

140

HELIX D -

- -

- -

>I

I

<-

--

I

<-

--

--

- HELIX E

- -

- -

>I

15

0 16

0 I

I Human IFNa2

EIMRSFSLSTNLQESLRSKE

Human IFNP

EILRNFYFINRLTGYLRN--

Murine IFNP

EIFRNFLIIRRLTRNFQN--

II

I

I I

It

I

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150

160

- HELIX E

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Hum

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uman

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NB

541163

791161

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~~~~

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N. J. MURGOLO ET AL. 66

TABLE 11. Interhelical Angles*

MuIFNP Helical HuIFNa2 MuIFNP X-ray angle model model structure6

f l A C -168.09 - 168.09 -168.4 - 149.94 - 149.94 -154.4 flB:C

a B E -166.77 -166.77 - 160.2 %!A - 155.95 -155.95 -157.2 *Interhelical angles were calculated by the stepped-helix rnethod7l as implemented in the ribbons program package" and are defined as the arc-cosine of the dot-product of the two helix vectors and is negative if the rotation is in the clockwise direction as defined by P r e ~ n e l l . ~ ~

a Silicon Graphics Personal IRIS 4D/25 computer with the Sybyl program.

Molecular Modeling of HuIFNa2 A primary sequence alignment between HuIFNa2

and MuIFNP based upon the algorithm of Needlman and Wunsch" is provided in Table I. HuIFNa2 was modeled as a single domain structure with 5 helices [residues 6-23 (A), 53-68 (B), 80-91 (C), 116-133 (D), and 141-156 (E)]. The homology model for HuIFNa2 was constructed from the determined MuIFNP structure through use of the Biopolymer module of the Sybyl program." Insertions and dele- tions were included in the model through use of SybyYBiopolymer. The overall number of insertions and deletions was minimal (8 of 165 residues) and involved mainly dipeptides in loops or a t the C-ter- minus of the MuIFNP structure (see Table I). Inser- tions were required at MuIFNP residues 27-28 (AB loop), 47-48 (AB loop), 103 (CD loop), and after 161 (C-terminus). A single deletion was required a t MuIFNp 44 (AB loop). The deletion and insertions required repair of the local backbone conformation by minimization. These insertions and deletions are rather short and did not alter the helix bundle core (see Table 11). The difference in AB loop length be- tween HuIFNa2 and MuIFNP is partly responsible for the conformation difference of the AB loop (see Fig. 2 and Discussion below). The HuIFNa2 model was refined by 500 cycles of constrained and 500 cycles of free minimization with IMPACT in a man- ner similar to that employed for MuIFNp. Helical constraints were again incorporated in the con- strained minimization, as well as two NOE-type constraints of 2.09 2 0.25 A used to form HuIFNa2's two disulfide bonds. Inclusion of the C29-Cl38 di- sulfide and the insertions and deletions at the AB loop, which contains C29, are likely responsible for the difference in conformation in this region be- tween the HuIFNa2 and MuIFNP models. Since the backbone Ca distance between disulfide paired C1- C98 was noted as 12 A in the unrefined model, a closer distance of approach of these residues was re- quired to form the disulfide bond.

AMCA-NHS Lysine Modification of HuIFNa2

AMCA-NHS (0.05 ml, 5.0 mg/ml) dissolved in dimethyl sulfoxide was added to HuIFNa2 (1.0 ml; 1.9 mglml, Schering-Plough Research Institute/Bio- technology-Bioisolation) dissolved in 0.1 M sodium phosphate, pH 8.4. After a 1 hr incubation at 22"C, the conjugate was evaluated for degree of AMCA modification by measuring the change in absor- bance at 280 and 345 nm.20 The data indicated there were 3.7 AMCA modified residues per mole of HuIFNa2. Given the molar excess of 7.7:l AMCA- NHSHuIFNa2, the corresponding efficiency of con- jugation was 48%. The AMCA modified HuIFNa2 was purified by reverse-phase high-performance liquid chromatography. The reaction mixture de- scribed above was chromatographed on a C, wide- pore (5 pm) column (4.6 x 30 cm, Rainin, Woburn, MA) using a 0 to 90% linear gradient of acetonitrile, 0.1% aqueous TFA (trifluoroacetic acid) over a 50- min period. The major UV absorbing peak was col- lected and lyophilized to an amorphous solid. This material was used for mass spectral analysis.

Trypsin Digestion of HuIFNa2 Recombinant HuIFNa2 and AMCA modified

HuIFNa2 were digested with trypsin for 16 hr at 37°C at a substrate:enzyme ratio of 50:l (w/w) in 1% (w/v) ammonium bicarbonate buffer adjusted to a pH of 8.4.

Plasma Desorption Mass Spectrometric Analysis of Trypsin Digests of HuIFNa2

Plasma desorption mass spectra were obtained on a BIOION 20 Californium-252 plasma desorption time-of-flight mass spectrometer using an acceler- ating voltage of 18 kV.21,22 Protein digest mixtures (400 pmol-1 nmol), in lyophylized form, were dissolved in a 1:2 mixture of ethanol:O.l% aqueous TFA, and then applied on a nitrocellulose (NO- coated aluminum foil and spin-dried prior to their analysis. Further thorough rinsing of the NC- adsorbed peptide mixture with 0.1% TFA or deionized water enhanced the signals of the higher mass peptide fragments.23 For molecular weight determination experiments the protein sample (1-2 nmol) was dissolved in a 1:2 mixture of ethanol: 0.1% aqueous TFA, applied onto an NC-coated aluminum foil and spin-dried prior to its insertion into the mass spectrometer. The time-of-flight spectra were acquired over a 1-3 hr period and then converted to mass spectra using the centroids for H' and NO+ as calibration peaks. The use of mass spectrometry for molecular weight determination and peptide mapping of HuIFNa2 has been previ- ously d e s ~ r i b e d . ~ ~ . ~ ~

HOMOLOGY MODEL OF HuIFNa2 67

Fig. 2. Stereo overlay of HulFNa2 and MulFNp models. Backbone trace diagram, with HulFNa2 model in cyan and MulFNp model in red. Every tenth residue is labeled. In the HulFNa2 model, disulfide side chains are added in yellow. The C294138 disulfide is at the top of the drawing at the putative active site region and the Cl-C98 disulfide is at the bottom. The N- and C-termini are located at the lower left. Largest deviation in overlap occurs in the AB loop (upper left; see text).

Ion-Spray Mass Spectrometric Analysis of Trypsin Digests of HuIFNd

The ion-spray spectra were recorded on a Sciex API I11 triple-quadrupole mass spectrometer equipped with an atmospheric pressure ion source and an ion-spray interfaceaZ5 Protein samples (5 mg/ml in 5050 methano1:water containing 0.1% TFA) were infused at 2 pYmin through the ion-spray interface. The final spectrum was an averaged sum of 10-20 scans from a mass to charge ratio (rnlz) of 1000 to 2400 at a scan rate of 3 s/scan.

RESULTS AND DISCUSSION MuIFNP

The refined model for MuIFNP generated using the STEREO program is consistent with the re- ported X-ray ~tructure.',~ The interhelical angles of the antiparallel four-helix bundle formed by helices A,B,C, and E in both the HuIFNa2 and MuIFNP models are consistent with the values observed in the crystal structure of MuIFNP (see Table II).6 Analysis of the solvent accessible surface areaz6 for the MuIFNP model using IMPACT gave a solvation free energy (AGSFE, free energy of the folded state with respect to the unfolded state) similar to the expected value for a monomeric protein containing 161 residue^.'^ There were no buried D, E, K, or R residues that did not participate in H-bonds or ion pairs. A detailed analysis of charged residues

showed that the side chains of E41, E109, and R147 were exposed. Ion pairs were observed for K16-E86, E57-Kl18, E100-K101, and E104-R105. Hydrogen bonding was observed or conceivable for K4S49, Q7-ElO (i to i + 31, N23-K27, Q60-K95, S71-R142, and E144-N148 (i to i + 4).

A helical wheel diagram of MuIFNp (Fig. 1) illus- trates several important structural features includ- ing the location of buried charged residues that par- ticipate in hydrogen bonding or ion-pair interactions (E10, E86, R142, and E144). Charged residues that do not participate in ion pairs or hydrogen bonds are seen to be externally localized in the figure. In ad- dition, this figure demonstrates that there are con- siderable hydrophobic interactions within the heli- cal core and that C17 is buried in the hydrophobic core. The amphipathic nature of helix A as shown in Figure 1 strongly suggests that the orientation pre- sented here, which includes the two amino acid ro- tation, is correct since the hydrophobic residues are buried in the core and the majority of charged resi- dues are exposed.

The reasonably compact structure and absence of buried charged residues which do not participate in stabilizing interactions are consistent with proper- ties expected for correctly folded protein model^.^^^^^ Additionally, the apolar to polar ratio of solvent- accessible surface areas was calculated as 1.616, suggesting that the overall folding pattern was cor- rect. Values of 1.769 for hemerythrin (correctly folded) and 2.50 for antibody variable light chain

68 N. J. MURGOLO ET AL.

Fig. 3. Stereodiagram of putative active site region. Backbone trace diagram of HuIFNaP. Constraining disulfide bond Cys-29- Cys-138 side chain atoms are indicated in yellow. AB, BC, and DE

domain forced onto a hemerythrin helix-bundle tem- plate (incorrectly folded) have previously been de- scribed.29 It is interesting to note that the presented orientation of helix A in the MuIFNp model results in nearly complete solvent inaccessibility of C17 (Fig. 1). Modification experiments of IFNP have sug- gested that C17 is m~difiable,~'-'~ but covalent modification may result in alteration of the confor- mation of MuIFNP, as reaction with DTNB caused a decrease in the activity of MuIFNP.~'

HuIFNa2

A stereodiagram of the model with disulfide posi- tions noted is provided in Figure 2. The final model incorporated the two known disulfide bonds located between Cl-C98 and C29-C138.33 The disulfides had SySy distances of 2.044 and 2.025 A, CQsy distances of 1.838, 1.828 and 1.817, 1.822 A, and CPSySy-Cp torsional angles of 114.5 and 129.0" for Cl-C98 and C29-Cl38 disulfides, respectively. The a-helical content of 48.5% (80/165 residues) was consistent with prior estimates determined by Ra- man and circular dichroism ~ t u d i e s . ' ~ . ~ ~ Tryptophan solvent accessibilities were noted as 21.4 and 1.6% for residues W76 and W140, respectively, and nei- ther side chain N d was exposed. It had been previ- ously noted that a t least one of the two tryptophans must be in a rigid apolar core region of the molecule based on circular dichroism and fluorescence stud- ies.15,34

The HuIFNa2 model is reasonably compact, as the AGSFE26 calculated with the IMPACT program was comparable to that expected for a monomeric protein with 165 residue^.'^ There were no buried D, E, K, or R residues that did not participate in H-bonds or ion pairs. Analysis of all charged residues showed sidechain exposure of E42 and D114. Ion pairs were observed for R22-El41-Rl44 (i to i + 31, E58-Kl12, K70-E71, and E146-R149 (i to i+3). Hydrogen bonding was observed or conceivable for R12-Ql58, K31-backbone A139 carbonyl, Q46-E51, QlOl- E107, and backbone R33 carbonyl-K133 (see Fig. 3).

loops are labeled. Side chain atoms of K31 and KI33 and their corresponding backbone carbonyl hydrogen bonding partners A139 and R33 are indicated.

The hydrogen bonding of K31 and K133 appeared to be a feature of the structural support of AB and DE loops in the HuIFNa2 model. AMCA-NHS modifica- tion was possible to only a limited extent at these two residues which may reflect some flexibility in this region of the structure (see Table 111 and discus- sion below).

A helical wheel diagram (Fig. 1) illustrates the considerable hydrophobic interactions within the helical protein core. This figure also shows the loca- tion of buried charged residues participating in hy- drogen bonding or ion-pair interactions (R12, R22, and R144) as well as exposed charged residues that do not participate in ion pairs or hydrogen bonds.

The ratio of apolar to polar side chain solvent ac- cessible surface area was calculated as 1.768. This result taken together with the reasonably compact single domain structure and participation of all bur- ied charged residues in favorable interactions sug- gests the three-dimensional structure presented here for HuIFNa2 closely resembles the native s t ru~ture . '~ A comparison of the MuIFNp and HuIFNa2 models showed backbone rms deviations of 0.807 A in helical regions and 3.100 A overall. The largest deviations occurred in the AB loop, un- doubtedly due to the introduction of the C29-C138 disulfide bond and the shorter AB loop length in MuIFNp. Residue C29 is in the AB loop. That the AB loop conformation differences may affect the rel- ative conformations of K31 and K133 in relation to the AB loop cannot be excluded. Stereographic views of overlapped MuIFNp and HuIFNa2 models are provided in Figure 2. The positions of K31 and K133 with respect to the putative active site loops are shown in Figure 3.

Lysine Accessibility of HuIFNa2

Prior chemical modification experiments with flu- orescamine have demonstrated the functional im- portance of accessible amino groups of IFNcY.~' ,~~ The AMCA-NHS lysine modification agent was em- ployed to elucidate buried lysines that were pro-

69 HOMOLOGY MODEL OF HuIFNa2

TABLE 111. PDMS Analysis of the Tryptic Digest of HuIFNa2

Unmodified AMCA

Peptide Position Sequence Tl 1-12 CDLPQTHSLGSR T1,io 1-12 and 84-112 Tl-S-S-Tlo T'1,lo 1-12 and 71-112 T1-S-S-Tg-10 TZ 13 R

T4 23 R T3 14-22 TLMLLAQMR

T5 24-31 ISLFSCLK T6 32-33 DR T,,,, 24-31 and 135-144 T5S-S-T,, TI5,,, 24-33 and 135-144 T5-5-S-T1, T"5,17 24-33 and 134-144 T5-&%3-T16-17 T"'5,17 24-33 and 132-144 T5-&+%T15-17 T, 34-49 HDFGFPQEEFGNQFQK T,, 32-49 DRHDFGFPQEEFGNQFQK T,-8 34-70 T7-8 T6-8 32-70 T6-8 T8 50-70 AETIPVLHEMIQQIFNLFSTK T's 50-67 AETIPVLHEMIQQIFNLF T9 71-83 DSSAAWDETLLDK TI0 84-112 FYTELY QQLNDLEACVIQG

TI, 113-120 EDSILAVR VGVTETPLMK

T,, 121 K T,, 122-125 WQR T12-13 121-125 KYFQR TI4 126-131 ITLYLK T,, 132-133 EK

T,, 135-144 YSPCAWEWR TI8 145-149 AEIMR T,, 150-162 SFSLSTNLQESLR T,, 163-164 SK T". 165 E

T,, 134 K

Calculated MH+ 1314.5 4617.3 6049.8

175.2 1077.4 175.2 911.2 290.3

2119.5 2389.8 2518.0 2775.3 1956.1 2227.4 4398.0 4669.2 2460.9 2144.5 1451.5 3304.8

903.0 147.2 613.7 741.9 751.0 276.3 147.2

1210.4 619.8

1482.6 234.3 147.2

observed MH+

1314.2 4618.5 6050.5

1077.8

911.4t

2119.00

-

-

-

- 1955.0 2227.7

- 2461.6 2144.8 1451.6t

903.1

613.6 741.9 750.9

-

- 1209.6 619.4

1483.5

observed Modified MH+ residue

1313.8

6262.0

1077.4

* -

-

- - -

2118.7+ 2605.0t 2948.2t 3420.6t - -

4613.2 4884.5 2460.8 2143.7 -

903.1 - -

957.0 -

- 1210.3t 619.6

1483.1

K(83) K(83)

K(31)*

K(31)' K(3 1/134)* K(31/133/134)*

K(121)

K(133)* K(134)*

5

5

+Weak signal. *Partial modification. 'Unclear whether this residue is modified or not. *--signal was not observed (small peptide fragments with MWs < 400 Da were probably washed off during the NC sample prepa- ration).

tected from modification. Plasma desorption and ion-spray mass spectrometric analysis showed that a total of 4-5 of the 10 lysines were modified, in agreement with the spectrophotometric estimate of 3.7 lysines per HuIFNa2.

Mapping of modified sites was performed by plasma desorption mass spectrometric analysis of tryptic digests of unmodified and AMCA modified HuIFNa2 (see Table III).35,36 For a lysine residue to be considered AMCA modified two criteria were re- quired. First, this lysine-containing tryptic frag- ment detected in unmodified HuIFNa2 had to be absent in the tryptic digest spectrum of the AMCA modified HuIFNa2. Second, since modification of the lysine residues prevents cleavage by trypsin, new mass spectral signals corresponding to contig- uous tryptic fragments containing the AMCA-mod-

ified lysine had to be observed in the plasma desorp- tion mass spectrum of the AMCA-treated HuIFNa2 tryptic digest. This is illustrated in the identifica- tion of the modification sites at the lysine residues 49,83, and 121. All of these sites were found to be completely modified (see Table 111). The modification of K49 was demonstrated by the presence of the T,-, and T, tryptic fragments in the unmodified, but not in the modified, tryptic digest spectra. In their place, tandem fragments T6-8 and T,-s containing an AMCA group at K49 were observed only in the AMCA-NHS modified spectra. For residue K83, only the mass spectrum of the modified digest contained a signal corresponding to the disulfide-linked pep- tide TISS-Tg-,o at 6262.0 Da, thus indicating co- valent modification of K83 with AMCA (see Table 111); whereas signals corresponding to tryptic frag-

70 N. J. MURGOLO ET AL.

TABLE IV. Comparison of Lysine Solvent Accessibilities and Ability to Modify L y h e s

With AMCA-NHS*

PDMS*** indicates Modification Surface AMC A-NHS possible in area

Lysine modification? model? Nt;(A') 31 Partial No 1.48+ 49 Yes Yes 34.13 70 No No 26.93* 83 Yes Yes 34.92

112 No No 6.35' 121 Yes Yes 36.61 131 Unclear Yes 41.39 133 Partial No 15.39** 134 Partial Yes 44.34 164 Unclear Yes 43.79 *For chemical modification with AMCA-NHS, a lysine's NL nitrogen was expected to be substantially exposed. "he result- ing model of human IFNa2 was consistent with mass spectral data and presence of 6 modifiable lysines. Lysines 31 and 133 which were expected to be unm&iable with AMCA-NHS from the HulFNa2 model were slightly modified and may indicate flexibility in the active site (see Discussion and Table m). +Participates in H-bond to A139 backbone carbonyl in DE loop. *Participates in K70-E71 ion pair. sParticipates in E58-Kl12 ion pair. **Participates in H-bond to R33 backbone carbonyl in AB loop. ***PDMS Plasma Desorption Mass Spectrum (see Table III).

ments T, and TISS-Tlo were observed in the mass spectrum of the unmodified digest. Modification of K121 was demonstrated by the presence of the TI,-,, fragment without the AMCA group in the unmodified digest, and the T12-13 fragment with the AMCA group at K121 in the modified digest.

We found that K70 and K112 were unmodified, since tryptic fragments T, containing K70 and Tl- SS-T,-,, containing K112 were detected in both un- modified and AMCA modified digests (see Table 111).

The ability to modify K131 and K164 could not be assessed due to the absence of the T,, fragment (K164) in the unmodified HuIFNa2 trypsin digest spectra and absence of any T14-containing fragment (K131) in the AMCA modified spectra (Table III). Partial modification of K31, K133, and K134 was demonstrated by the limited ability of trypsin to cleave these residues (Table III).

Of the 10 lysines in HuIFNa2, the homology model suggested K31, K70, K112, and K133 would be protected from modification due to their low sol- vent accessibility (see Table IV). The inability of AMCA-NHS to modify K70 and K112 supports their solvent inaccessibility in the homology model. The partial modification of K31, K133, and K134, present in the AB and DE loops, may suggest vari- ability in the relative loop conformations (see dis- cussion below).

The presence of K31 at a position in the AB loop that does not appear critical to the core packing of

the HuIFNa2 model was supported by sequencing of electrophoretically resolved endoproteinase lys-C di- gestion fragment^.^' Digestion experiments showed that within 30 min this lysine was the primary cleavage site and far- W circular dichroism indi- cated that cleavage at K31 did not affect the second- ary structure of HuIFNa2 (data not shown). Further digestion led to cleavage at K70, probably due to protein denaturation. Digestion times up to 20 hr led to this additional cleavage at K70, K49, and K133 with structural perturbations equivalent to a-helical unfolding as observed in circular dichroism studies (data not shown).

Comparison of HuIFNd, MuIFNp Models With Known Properties

The predicted folding pattern for HuIFNa2 is con- sistent with known disulfide pairing information and tryptic maps (see below).93,3s*39 The difference in backbone orientation in the AB loops of HuIFNa2 and MuIFNp may reflect structural differences be- tween the two molecules required for disulfide bond formation. A similar difference in the AB loops of MuIFNp and HuIFNp has been previously de- scribed.6 The MuIFNp model is also consistent with formation of a disulfide bond between C31 and C141 in its homologous HuIFNp counterpart (within AE3 and DE loops). The inclusion of this disulfide in re- combinant murine IFNB has recently been shown to enhance its a~tivity.~'

The determined models for MuIFNP and HuIFNa2 are consistent with the location of the pu- tative active sites involving adjacent AB, BC, and DE loops (24-52,69-79, and 134-140 in HdFNa2; see Fig. 3). These residues, especially those in the AB and DE loops, are poorly conserved between MuIFNp and HuIFNa2 as would be expected for re- gions required to establish binding specificity (see Table I). Three domains of HuIFNa2 have been iden- tified that are critical for antiviral activity, induc- tion of 2',5'-oligoadenylate synthetase, and cell growth i n h i b i t i ~ n . ~ ~ The regions, located within 10- 35, 78-107, and 123-166, are at or near the three loops at the top of the model shown in Figure 2. Demonstration of the functional importance for di- sulfide C29-C138, which is proximal to the loop re- gion identified above, and the lack of functional im- portance for the distal disulfide C 1 4 9 8 is consistent with both the model and the predicted binding epitopes (see Figs. 2 and 3).16 The homolo- gous disulfide bond in HuIFNp, which is similarly predicted to lie in the Al3/DE loop functional epitope, has also been shown to be required?'

The left-handed antiparallel orientation of the A,B,C, and E four helix bundle structure presented in the HuIFNa2 and MuIFNp models may represent an evolutionary conserved feature of cytokines and

HOMOLOGY MODEL OF HuIFNa2 71

growth factors. This structure has been previously observed in the three-dimensional structures of granulocyte-macrophage colony-stimulating fac- t ~ r : ~ , ~ in t e r l e~k in -4 ,4~-~~ interferon-y,& and growth h0rmone.4~*" The binding of HuIFNa2 to its receptor may be similar to that of the human growth hormonelreceptor complex5' as the regions identi- fied by previous mapping experiments may repre- sent only a portion of HuIFNaa's binding site. Such a model would suggest helices A and E and the AB loop comprise site 1 and helices A and C comprise site 2.

Biological Mapping Studies

Site-directed mutagenesis and peptides

Site-directed mutagenesis experiments on HuIFNa4 have shown the importance of AB loop residues L30, R33, and F36 for expression of antivi- ral and antiproliferative a~tivities.'~ Interestingly, the mutation of K31R and K31N did not affect an- tiviral activity, but K31E lowered antiviral activity on WISH cells to 16% that of wild type.51 Analysis of the HuIFNa2 structural model suggests that K31 interacts with the backbone of the DE loop at resi- due A139 (see Fig. 3). This interaction may play some role in defining the active site conformation of a presumably flexible AB loop and limit the acces- sibility of K31, as this lysine could only be modified to a small extent with AMCA-NHS as detected by mass spectral analysis of tryptic digests of HuIFNaZb (see Table 111). The observed preserva- tion of activity with the mutations K31R and K31N is consistent with the HuIFNa2 model, as they would be expected to continue the support of the AB loop through hydrogen bonding to the backbone car- bony1 at A139 (see Fig. 3). The inactive K31E mu- tant may disrupt this interaction and affect the structure of this portion of the active site. The exact orientation of K31 will undoubtedly require a de- tailed structural analysis by X-ray crystallography. Similar AB loop mutation studies have stressed the importance of R33.52 Point mutations near the DE loop of HuIFNa2, which introduced residues found in HuIFNal, were noted to impart murine receptor binding." Point mutations in the 121-136 (DE loop region) of HuIFNa, especially a t position 125, have been shown to alter antiviral a~tivity. '~

Several studies have demonstrated mutation of buried helix D residues Y122 and 123 of HulFNa to anything other than aromatic residues leads to loss of activity (i.e., Yl22K,D,S,A,G; F123S are inactive while Y122F,W are These changes would be expected to structurally disrupt the inner core of HuIFNa2 based upon our present model (Fig. 1).

Deletion of 13 C-terminal residues or 4 N-termi- nal helix A residues, which are at the opposite side

of the model from the AB, BC, and DE loops, does not affect bioactivity of HuIFNa.15

Chimeric interferons A number of chimeric molecules have been con-

structed to map the biologically important sites of HuIFNa. Binding studies using murinehuman IFNa hybrids suggested that the binding site was discontinuous, involving some or all of regions 1-12 (helix A), 25-38 (AB loop), 70-74 (BC loop), or 103- 113 (CD loop). The above model would include two of these regions as binding e p i t o p e ~ . ~ ~ Studies employ- ing bovinehuman chimeras have localized a recep- tor binding region in the AB loop including at least 5 residues.56

Antibody epitope mapping Several neutralizing antibodies have been epitope

mapped for HuIFNa and MuIFNp. Neutralizing an- tibodies raised against HuIFNa2 have been mapped to either the A helix residues 10-11 or a region in the BC loop.57*58 Other mapping experiments sug- gested the importance of these regions as well as the AB Neutralizing antibodies to HuIFNp have shown the importance of residues within the BC loop epitope including residues 41-43, and 46.60,61 The lack of importance of the last 16 residues for recep- tor binding of HuIFNa has been implied by devel- opment of a nonneutralizing monoclonal that recog- nizes cell-surface bound IFNa.62 Mapping of two antibodies directed to IFNa2c has also demonstrated the functional importance of regions 36-41 and 112-148 (including AB and DE loops).63

Alternative modele of HuIFNa Several previous conceptual models have been

proposed for HuIFNa2. Early secondary structural prediction algorithms suggested that HuIFNa2 would have a helix bundle ~ t r u c t u r e . ~ ~ ~ ~ ~ * ~ One of the earliest proposals was a 4-helix right-handed bundle (helices at residues 15-27, 57-72, 81-106, and 143-154).65 In this model, for which coordinates have been presented, the connections between heli- ces a t residues 28-56 and 107-142 were proposed as a two-stranded antiparallel p sheet. While this model is consistent with a single domain and highly helical structure, it contains two extremely large AB and CD loops. In addition, to provide a right- handed orientation, the N-terminus is proposed to lie parallel along the helix bundle to allow for for- mation of the C 1 4 9 8 disulfide, whose pairing would be accommodated in a more compact struc- ture if helices A and C were antiparallel, not paral- lel as proposed in that model.65 A later model pro- posed a two-domain structure (a right-handed 4-helix bundle first domain, with either three heli- ces or two helices packed against a p sheet face for the second domain), which although highly helical, is inconsistent with biological activity mapping and

72 N. J. MURGOLO ET AL.

proteolytic digestion experiments which have not to date indicated two distinct

Recent fluorescence polarization experiments suggested IFNa has a two domain structure with an axial ratio (a:b) of 4:1.67 However, prior sedimenta- tion velocity measurements by analytical ultracen- trifugation on monomeric HuIFNa suggested a more compact single domain s t r u c t ~ r e . ~ ~ * ~ ~ The model for HuIFNa2 proposed in our study has an axial ratio of 1 2 1 , measured from the smallest possible solute- enclosed box (44.7 x 48.1 x 55.9 A). Using these values, a theoretical sedimentation coefficient for a rigid, nonhydrated sphere was calculated as 1.82 S,68 in agreement with the previously observed sed- imentation coefficient of 1.95 S for H d ~ N a 2 . ~ ~ Based upon chimeric mapping, another model has been proposed containing 6 helices (residues 13-24, 39-52,53-69,75-102,114-133, and 140-162)55 in an antiparallel arrangement with distinct spatial lo- cations of putative binding epitopes. The cylindrical helix HuIFNa2 model proposed by Zav’yalov et al.70 and most recently by Fish74 is similar in helical as- signments to the model proposed here. However, our model represents the first atomic coordinate struc- ture for HuIFNa2 that is consistent with its known biochemical and biophysical features.

ACKNOWLEDGMENTS We wish to acknowledge Mr. Stanley Mittelman

for circular dichroism analysis, and Drs. Stephen Tindall and Hung Le for help in preparation of the manuscript. Coordinates of model structures will be submitted to the Brookhaven Protein Databank.

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