Inducible Nitric Oxide Synthase Requires Both the Canonical Calmodulin-binding Domain and Additional Sequences in Order to Bind Calmodulin and Produce Nitric Oxide in the Absence of Free Ca2+

Abstract

All three mammalian isoforms of nitric oxide synthase (NOS) must bind calmodulin (CaM) for enzymatic activity. Only NOS2 (the inducible isoform, iNOS) does so at the low levels of free Ca2+ in resting cells and when almost all Ca2+ is chelated in cell-free preparations. To test directly whether the predicted CaM-binding region of mouse NOS2 accounts for its Ca2+ independence, we prepared chimeric NOS's in which mouse NOS2 residues 503-532 were reciprocally exchanged with the corresponding residues 725-754 of rat NOS1 (neuronal NOS). Unlike either parent, both chimeras required an intermediate level of free Ca2+ to bind CaM and generate NO. In cell lysates, the concentration of Ca2+ necessary for half-maximal activity (EC50) was approximately 0 for NOS2, 200-300 n for NOS1, and 7-10 n for the chimeras. Results were similar when the region exchanged was enlarged by 7-8 residues toward the amino terminus. In contrast, when the carboxyl-terminal half of NOS2 (residues 454-1144) was replaced with that of NOS1 (residues 675-1429), the resulting chimera resembled NOS1 (EC50, 200-300 n free Ca2+). Truncation analysis suggested that NOS2 residues within the sequence 484-726 were required for Ca2+-independent CaM-binding. Thus, both the canonical CaM-binding domain and additional residues within the region 484-726 are necessary for NOS2's ability to bind CaM and produce NO when Ca2+ levels approach zero.

Nitric-oxide Synthase (NOS) Reductase Domain Models Suggest a New Control Element in Endothelial NOS That Attenuates Calmodulin-dependent Activity

“…6). This is consistent with other observations that Ca 2ϩ dependence of NO activity, and therefore CaM binding, is determined by multiple interactions in the CaM binding region (30,71,72). Progressive replacement of the eNOS reductase domain segments with sequences from iNOS would eventually lead to the E/I chimera, which indeed exhibited virtual Ca 2ϩ independence (22).…”

“…The Ca 2ϩ dependence of NOS activity could also be reduced in various nNOS chimeras that contain either the iNOS heme or reductase domain along with the iNOS CaM recognition helix, but only the iNOS reductase-containing chimera demonstrated full Ca 2ϩ independence (72). iNOS truncation mutants with successive N-or C-terminal deletions demonstrated a similar requirement of iNOS residues between 490 and 732 for Ca 2ϩ -independent CaM binding (71). This evidence has implications for the work presented here, because residues 490 -732 include the CaM recognition helix, the FMN, and the CD-1 subdomains, all of which are within direct contact distance of either the AI element or the CD2A loop (Fig.…”

“…Circular dichroism spectroscopic observations show that the CaM recognition element from iNOS (residues 509 -535) actually binds to apo-CaM in a type II ␤-turn conformation, and its conformation becomes helical when CaM is Ca 2ϩ -bound (70); therefore, iNOS has at least two modes in which to bind CaM. NOS chimeras that swap CaM recognition helices between isoforms have the same Ca 2ϩ dependence of CaM binding, intermediate between cNOSs and iNOS and only slightly reduced overall activity when compared with the parent isoform (30,71,72). Thus, only part of the Ca 2ϩ dependence of CaM binding is the result of the canonical CaM binding helix itself.…”

Structural basis for endothelial nitric oxide synthase binding to calmodulin

“…Synthetic peptides corresponding to the iNOS CaM-binding region generally have higher af®nity (<10 ±10 ±10 ±9 M) for Ca 2+ -loaded CaM than do those derived from the eNOS or nNOS CaM-binding region (10 ±9 ±10 ±8 M) (Vorherr et al, 1993;Zhang and Vogel, 1994;Anagli et al, 1995;Venema et al, 1996;Zoche et al, 1996;Matsubara et al, 1997;Yuan et al, 1998;Leclerc et al, 1999;Censarek et al, 2002). Chimeric nNOS or eNOS containing the iNOS CaM-binding region shows the decreased Ca 2+ sensitivity (Ruan et al, 1996;Venema et al, 1996), suggesting that the CaM-binding region itself contributes to the Ca 2+ -independent iNOS activation by CaM. Furthermore, several studies report that synthetic peptides corresponding to the iNOS CaM-binding region, unlike the eNOS and nNOS counterparts, form complexes with CaM in the absence of Ca 2+ (Anagli et al, 1995;Zoche et al, 1996;Matsubara et al, 1997;Yuan et al, 1998).…”

“…Recent studies suggest that the iNOS CaM-binding region is necessary, but not suf®cient for Ca 2+ -independent iNOS activity (Ruan et al, 1996;Venema et al, 1996;Lee and Stull, 1998). Synthetic peptides corresponding to the iNOS CaM-binding region generally have higher af®nity (<10 ±10 ±10 ±9 M) for Ca 2+ -loaded CaM than do those derived from the eNOS or nNOS CaM-binding region (10 ±9 ±10 ±8 M) (Vorherr et al, 1993;Zhang and Vogel, 1994;Anagli et al, 1995;Venema et al, 1996;Zoche et al, 1996;Matsubara et al, 1997;Yuan et al, 1998;Leclerc et al, 1999;Censarek et al, 2002).…”