The rice RLF structure confirms N-terminal helix formation
involving the Trp120 side chain.
Trp120 in RLF (corresponding to Trp37 in Ncb5or) is part of an
11-residue α-helix, designated α1
(S116QMDWLKLTRT126), that packs
against the side of the heme-binding pocket defined by helices α3 and α6
(Figure 7 ). In this and subsequent sections, all corresponding
residues in Ncb5or are listed in parentheses. A 17-residue stretch
connects α1 to α2, the first element of defined secondary structure in
the “classic” b5 core (α2 is the only helix in the b5 core that does
not comprise part of the heme binding pocket). The corresponding stretch
in Ncb5or is two residues shorter, with the gap appearing close to the
junction with α2. There are multiple conserved residues in the RLF and
Ncb5or loops, and in the RLF structure several of them exhibit specific
backbone and side chain interactions both within the loop and with the
b5 core. These include two three-residue turns featuring a single
backbone hydrogen bond, the first between Asp129 and Gly132 (Asp46 and
Gly49), and the second between Leu133 and Gln136 (Leu 50 and Arg53). The
Asp129 (Asp46) side chain in the first turn extends this motif via
hydrogen bonds to the backbone amide N-H groups of Leu133 and Lys134
(Leu50 and Lys51), residues which can be considered to represent the
transition between N-term and the b5 core. The side chain of Leu133
(Leu50) is buried and engages in hydrophobic interactions with several
nearby residues, namely Cβ of Ala131 (Thr48), Cβ of Gln136 (Arg53), and
Cα of Asn138 (Ile55). It also packs against the side chain of Val211
(Val126) which is in strand β4 of the b5 core. All the residues in this
hydrogen bonded array other than solvent exposed Gln136 (Arg53) are
conserved in RLF and Ncb5or proteins. Likely because of these
interactions, this polypeptide segment is well-ordered. Packing between
the N-terminal helix and the b5 core involves primarily hydrophobic
interactions and does not noticeably impact the b5 core fold. Consistent
with the results of far-UV CD studies described above, the Trp120 side
chain is situated in an environment with well-defined tertiary structure
that includes hydrophobic packing interactions with the side chains of
four residues in α3 and α6, each corresponding to a residue that is
highly conserved in Ncb5or: Tyr170 and Phe173 in α3 (Tyr85 and Tyr88);
and Leu206 and Cys209 in α6 (Leu121 and Cys124). The Trp120 side chain
engages in additional hydrophobic interactions with the nearby side
chains of Leu121 and Thr124 in α1 (Ile38 and Thr41) and with the side
chain of Leu130 (Leu47), located in the first 3-residue turn in the
17-residue loop connecting α1 to α2.
It is worth noting that the interaction between the Trp120 and Cys209
side chains (Trp37 and Cys124) involves the cysteine thiol (SH group)
packing against the tryptophan pi system. Such interactions are
relatively common and are thought to play a stabilizing
role.52,
53 Both residues are invariant in
Ncb5or proteins.
The Trp120 (Trp37) side chain has one additional contact, a hydrogen
bond between its N-H group and the backbone carbonyl of Leu205 in α6
(Met120). As noted in the previous section, the backbone carbonyl of
that residue does not form an intra-helix H-bond due to a kink in α6.
The present results suggest that the α6 kink may have evolved to serve a
distinct functional role in these proteins.
The only other polar contacts between α1 and the b5 core comprise a
network of hydrogen bonds involving the side chains of the first two α1
residues Ser116 and Gln117 (Ser31 and Leu32), the phenolic side chain of
Tyr170 in α3 (Tyr85), and the backbone carbonyl of Phe173 in α3 (Tyr88).
While this network of hydrogen bonds may be important in stabilizing α1,
it is noteworthy that the polar OH group of the Tyr170 side chain also
makes van der Waals contact with one of the heme meso positions.
This counterintuitive positioning of a polar group suggests a potential
functional role. An intriguing possibility is that oxidation of ferrous
heme to ferric heme during reduction of the downstream substrate is
accompanied by deprotonation of the phenolic group. This could
conceivably facilitate a coupled electron/proton transfer process during
substrate reduction. Alternatively, it could allow for electrostatic
stabilization of the b5 domain when heme is in the ferric oxidation
state and bears a formal positive charge.