Chapter 22
Solution Structure of Sea Anemone Toxins by NMR Spectroscopy 1,3
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N. Vasant Kumar , Joseph H. B. Pease , Hugues Schweitz , and David E. Wemmer Downloaded via UNIV OF MINNESOTA on July 10, 2018 at 14:18:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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Department of Chemistry, University of California at Berkeley, and Chemical Biodynamics Division, Lawrence Berkeley Laboratory, Berkeley, CA 94720 Centre de Biochimie du Centre National de la Recherche Scientifique, Faculté des Sciences Université de Nice, Parc Valrose, 06034 Nice Cedex, France 2
Numerous organisms, both marine and terrestrial, produce protein toxins. These are typically relatively small, and rich in disulfide crosslinks. Since they are often difficult to crystallize, relatively few structures from this class of proteins are known. In the past five years two dimensional NMR methods have developed to the point where they can be used to determine the solution structures of small proteins and nucleic acids. We have analyzed the structures of toxins II and III of Radianthus paumotensis using this approach. We find that the dominant structure is ß - s h e e t , with the strands connected by loops of irregular structure. Most of the residues which have been determined to be important for toxicity are contained in one of the loops. The general methods used for structure analysis will be described, and the structures of the toxins RpII and RpIII will be discussed and compared with homologous toxins from other anemone species.
Sea anemones use a variety of small proteins (ca. 5 kD) as part of their powerful defense and feeding systems (7). These proteins function as neurotoxins and/or cardiotoxins by binding to sodium channels and thereby altering ion-conducting characteristics of the channels. Binding of toxin slows down the inactivation process of the channels and hence prolongs the duration of action potential (2). Toxicity of these proteins depends on the type of organism against which they act. For example, some toxins are very potent against crustacean and mammalian channels whereas some are effective only on crustaceans. Tetrodotoxin (TTX)-resistant channels in general seem to have higher affinity than the TTX-sensitive channels for these toxins. In fact, such differences in affinities of these toxins for different channels have been exploited to find the subtypes of sodium channels.
Current address: SmithKline & French, Physical and Structural Chemistry, Box 1539, King Prussia, PA 19406-0939
0097-6156/90/0418-0290S06.00/0 o 1990 American Chemical Society
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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Solution Structure of Sea Anemone Toxins
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Despite the vast amount o f data o n the pharmacological properties, very little about the conformation o f the proteins has been k n o w n u n t i l recent N M R studies. 2 D - N M R results have provided detailed information about the secondary structure o f several related anemone toxins. A T X I from Anemonia sulcata (3,4) and A P - A from Anthopleura xanthogrammica (5,6) have been studied by G o o l e y and N o r t o n , and more recently W i d m e r et a l . have further purified the A. sulcata toxins and obtained complete sequence specific assignments for A T X l a (7). O u r laboratory, o n the other hand, has studied the structures o f R p I I and R p I I I from Radianthus pawnotensis (8). Schweitz et a l . purified four related toxins ( R p l , R p I I , R p I I I , and R p I V ) from sea anemone Radianthus pawnotensis ( R p ) and studied their pharmacological properties (9). D u r i n g the course o f initial N M R studies, the reported sequence o f R p I I was found to have errors, and was redetermined (8). Subsequently M e t r i o n e et a l . determined the sequence o f R p I I I as well (10). T h e other two R p toxin sequences are yet to be determined. Sequences o f the R p , and several other sea anemone toxins, are shown i n Table I. W e have used a two letter code to denote the species consistently and this notation differs from the earlier designations o f N o r t o n and W u t h r i c h groups. In o u r notation, A s l a and A x I correspond to A T X l a and A P - A , respectively. F r o m alignment o f the cystines i n these sequences, it is clear that R p toxins have three disulfide bonds, as do the other toxins. T h e sequences o f R p I I and R p I I I are very similar to o n e another, but differ m o r e significantly from the other A s and A x toxins. Schweitz et a l . showed that these toxins are functionally similar to other sea anemone toxins, that is, toxins slow down the inactivation process o f sodium channels. However, R p toxins do not affect the binding o f A. sulcata toxins to the sodium channels, and it is also interesting that the R p toxins compete with the scorpion toxin A a l l from Androctonus australis for binding sites o n sodium channels, even though their primary sequences appear completely different. They further demonstrated that R p toxins are immunologically distinct from A. sulcata ( A s ) o r A. xanthgrammica ( A x ) toxins. A n t i b o d i e s raised against R p I I I recognize a l l other R p toxins but not AsII, A s V , A x l , A x i l . Conversely, antibodies to A s I I o r A s V do not recognize any o f the R p toxins. W i t h o u t a knowledge o f the structure o f these proteins it is difficult to interpret these results. W e have undertaken the study o f the structure o f R p toxins by 2 D - N M R and distance geometry techniques to try to understand these functional relationships. H e r e we review the underlying methods briefly, discuss the structures o f these toxins, and compare them with what is k n o w n about the other related toxins. 2 D - N M R Techniques T h e first step i n determination o f a structure by N M R spectroscopy involves assignment o f individual p r o t o n resonances. Development o f high-field spectrometers and the use o f a second dimension ( 2 D - N M R ) along with isotopic substitution (11) and sophisticated pulse sequences (12) make it possible to almost completely assign the p r o t o n spectrum o f proteins o f about 15 k D molecular weight (13-17). S o m e 2 D pulse sequences c o m m o n l y used i n the study o f macromolecules are shown i n F i g ure 1. A l l these experiments involve at least three distinct time periods: preparation (tp), evolution ( t l ) , and detection (t2); these periods are usually separated by rf pulses. S o m e experiments (e.g., N O E S Y , R E L A Y ) further contain an additional " M i x i n g " period, t m , between the evolution and detection periods. T h e spin system is perturbed by rf pulses during the preparation period to create the desired coherences which are then allowed to evolve during the time t l ,
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990. -GNCKCDDEGPNVRTAPLTGYVDLGY—CNEGWEKCASYYSPIAECCRKKK
Radianthus paumotensis III (RP III)
GAACLCKSDGPNTRGNSMSGTIWVF—GCPSGWNNCEGRA-IIGYCCKQ GAPCLCKSDGPNTRGNSMSGTIWVF—GCPSGWNNCEGRA-I IGYCCKQ GVPCLCDSDGPSVRGNTLSGIIWLA—GCPSGWHNCKKHGPTIGWCCKQ GVPCLCDSDGPSVRGNTLSGILWLA—GCPSGWHNCKKHKPTIGWCCK GVSCLCDSDGPSVRGNTLSGTLWLYPSGCPSGWHNCKAHGPTIGWCCKQ GVPCLCDSDGPRPRGNTLSGILWFYPSGCPSGWHNCKAHGPNIGWCCKK
Anemonia sulcata la (AS la)
Anemonia sulcata lb (AS lb)
Anemonia sulcata II (AS II)
Anemonia sulcata V (AS V)
Anthopleura xanthogrammica (AX I)
Anthopleura xanthogrammica (AX II)
Radianthus macrodactylus III (RM HI) -GNCKCDDEGPYVRTAPLTGYVDLGY—CNEGWEKCASYYSPIAECCRKKK
-ASCKCDDDGPDVRSATFTGTVDFWN—CNEGWEKCTAVYTPVASCCRKKK
Radianthus paumotensis II (RP II)
Sequences of Sea Anemone Toxins
Table I
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KUMAR ETAL.
Solution Structure of Sea Anemone Toxins
n/2
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COSY
t1
tp
n/2
DQFCOSY
tp
n/2
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n/2 RELAY tp
n n/2
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Figure 1. Pulse sequences o f some typical 2 D - N M R experiments. C O S Y = Correlation Spectroscopy D Q F C O S Y = Double Quantum Filtered C O S Y , R E L A Y = R E L A Y e d Magnetization Spectroscopy, and N O E S Y = Nuclear Overhauser Effect SpectroscopY.
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY
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frequency labeling them. T h e signal (or free i n d u c t i o n decay, F I D ) is detected during the t i m e t2 and it carries information about behavior o f spin system during the t i m e t l , a n d also about h o w the information was exchanged by spins during the mixing time. T h e e v o l u t i o n period t l is systematically incremented i n a 2D-experiment and the signals are recorded i n the form o f a time d o m a i n data matrix S ( t l , t 2 ) . T y p i cally, this matrix i n o u r experiments has the dimensions o f 512 points i n t l and 1024 i n t2. T h e frequency d o m a i n spectrum F ( w l , u)2) is derived from this data by successive F o u r i e r transformation w i t h respect to t2 and t l . 2D-spectra are usually represented by contour plots (Figures 2 - 4 ). Peaks with the same frequency i n b o t h the dimensions (a;l=u;2) are called the diagonal peaks and correspond to the resonances i n the I D - s p e c t r u m . I n cross peaks, for w h i c h 2, the intensity and structure contain important information about the t o p o l ogy and dynamics o f the spin system. F o r example, a cross peak at (CJ1,O;2) i n a C O S Y o r a m u l t i p l e quantum filtered C O S Y spectrum implies that the spin A with resonance frequency C J I and another spin B w i t h frequency u2 are coupled through , ) * R E L A Y spectrum with appropriate delay 2T i n contrast to the C O S Y cross peaks at ( ^ , ^ ) and ( ^ ^ ) * ^ ^ y °f P ^ depends o n the magnitude o f c o u p l i n g constants and J ™ i n a nonlinear manner. A s a result, it is difficult to obtain information about a l l the remote connectivities from a single R E L A Y experiment. A related experiment T O C S Y (Total C o r r e l a t i o n Spectroscopy) gives similar information and is relatively m o r e sensitive than the R E L A Y . O n the other hand, intensity o f cross peak i n a N O E S Y spectrum with a short mixing time is a measure o f internuclear distance (less than 4 A ) . It depends o n the correlation time r and varies as < r " > . It is positive for small molecules with short correlation time (a>r l ) and goes through zero for molecules w i t h r ^l. R e l a x a t i o n effects should be taken i n t o consideration for quantitative interpretation o f N O E intensities, however. n
A
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e
n
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c
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Sequence-Specific Assignments Systematic analysis o f interproton distances i n proteins l e d W u t h r i c h and colleagues to formulate the sequential assignment algorithm (18, 19). I n this method sets o f scalar c o u p l e d spins corresponding to the individual amino acids are identified using various correlation experiments ( C O S Y , R E L A Y , T O C S Y , etc.), and then sequence specific assignments are obtained by analyzing N O E cross peaks w h i c h correspond to neighbors i n the primary sequence. W e have assigned R p I I and R p I I I by basically following this procedure. T h e D Q F C O S Y spectrum o f R p I I i n D 0 is shown i n F i g u r e 2. E a c h cross peak i n this spectrum identifies a pair o f coupled spins o f the amino acid side chains. Since couplings are not propagated efficiently across amide bonds, a l l groups o f c o u p l e d spins occur w i t h i n individual amino acids. T h e chemical structure o f an amino acid side chain is reflected i n the characteristic c o u p l i n g network and chemical shifts (13). V a l i n e spin system ( C H - C H - ( C H ) ) is explicitly shown i n Figure 2 as an example. 2
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Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
22. KUMAR ETAL.
Solution Structure of Sea Anemone Toxins
Figure 2. D Q F C O S Y spectrum o f R p I I i n D 0 . Cross peaks corresponding to a-fi protons o f various spin systems are labeled. T h e valine spin systems are shown explicitly. 2
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY
0K46
0S14
o 0
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PPM Figure 3. Fingerprint region o f the C O S Y spectrum o f R p I I i n H ^ O showing the amide to a p r o t o n connectivities. Cross peaks are labeled w i t h the identity o f the amino acid i n the sequence from which they arise.
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990. 30
S3
ESS 40
KSSS
SSS
KS
KSS
EZ2
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ESS
NV\N\N E S S LVANVt
Figure 4. Summary o f the sequential N O E s observed i n R p I I . A bar connecting the residues means that the indicated type o f connectivity was observed between those residues.
ESS NNNVSN
H
N C N E G W E K C T A V Y T P V A S C C R K K K
LViVvVWi
A S C K C D D D G P D V R S A T F T G T V D F W
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It was straightforward to identify the spin systems o f four valines, five threonines, four alanines, three glycines, two glutamates, and A M X type residues (Asp, Cys, Phe, Tyr, T r p , and Ser) o f this protein from the C O S Y , R E L A Y , and N O E S Y spectra i n D J D solution. T h e R E L A Y spectrum was particularly useful i n identification o f V a l , A l a , Thr, and G l u spin systems. T o identify the resonances o f amide protons (which can exchange with solvent) additional experiments were carried out i n H 0 solution. T h e fingerprint region, corresponding to cross peaks between amide and alpha protons, is shown i n Figure 3. Degeneracies i n alpha p r o t o n chemical shifts were partly resolved by finding the A f¥ peaks corresponding to the given amide chemical shift i n the R E L A Y spectrum, and also by varying the experimental conditions such as temperature. Sequence-specific assignments were obtained by analysis o f the N O E S Y and C O S Y spectra obtained under the similar conditions i n H 0 . T h e three important classes o f N O E cross peaks for this purpose are designated d , d ^ , and d - In this notation, a d connectivity indicates an N O E crosspeak i n a short mixing time N O E S Y between X and Y protons belonging to neighboring residues i and i + 1 , and hence corresponding to a distance o f
